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GelRed™ and GelGreen™ troubleshooting (2)

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Many customers use GelRed™ or GelGreen™ precast gels for convenience. However, because GelRed™ and GelGreen™ are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed™ or GelGreen™ precast gels.

Tip #1: Load less DNA

Smearing and smiling in GelRed™ or GelGreen™ precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems.

Tip #2: Try the post-staining protocol

To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused. See the GelRed™ Product Information Sheet or GelGreen™ Product Information Sheet for detailed protocols.

Other tips to improve agarose gel resolution:

  • If you see DNA migration issues or smearing after post-staining with GelRed or GelGreen, then the problem is not caused by the nucleic acid dye. Avoid overfilling gel wells to prevent smearing of DNA down the surface of the gel.
  • Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
  • Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.

The dye may have precipitated out of solution.

  1. Heat GelRed™ solution to 45-50°C for two minutes and vortex to dissolve.
  2. Store dye at room temperature to avoid precipitation.

GelRed™ and GelGreen™ Nucleic Acid Gel Stains (36)

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The main difference between GelRed™ and GelGreen™ is their fluorescence excitation and emission wavelengths. GelRed™ has red fluorescence, similar to ethidium bromide. GelGreen™ has green fluorescence, similar to SYBR® Green or SYBR® Safe. Both dyes are compatible with standard UV transilluminators. GelGreen™ is also compatible with blue light transilluminators, which allow users to avoid exposing themselves and their DNA samples to ultraviolet radiation.

GelRed™ and GelGreen™ have higher sensitivity for double stranded nucleic acids compared to single stranded nucleic acids, but GelRed™ is more sensitive for staining single stranded nucleic acids than GelGreen™. GelRed™ is about twice as sensitive for double stranded nucleic acids compared to single-stranded nucleic acids, and about five times more sensitive than GelGreen™ for staining single stranded nucleic acids.

For more information about these products, please visit our DNA stains technology page.

View the GelRed™ Product Line

View the GelGreen™ Product Line

The water formulation is a newer and improved product compared to the stock in DMSO. We recommend using dyes in water to avoid the potential hazards of handling DMSO, which can be absorbed through the skin. We continue to offer dyes in DMSO because some users do not wish to alter their established laboratory protocols. Based on internal testing, both formulations perform similarly.

GelRed™ is compatible with a standard UV transilluminator (302 or 312 nm).
GelGreen™ has sufficient absorption between 250-300 nm and a strong absorption peak at around 500 nm. GelGreen™ is compatible with a 254 nm UV transilluminator or a gel reader with visible light excitation such as a Dark Reader or a 488 nm laser gel scanner.

Use the ethidium bromide filter for GelRed™; use a SYBR™ Green or yellow filter for GelGreen™. Alternatively, a long-pass yellow filter can be used with both GelRed™ and GelGreen™. Please review the emission spectra for GelRed™ and GelGreen™ for more specific wavelengths.

In AMES and related tests, GelRed™ and GelGreen™ were shown to be much safer alternatives to EtBr and SYBR dyes. Nevertheless, please exercise safe laboratory practices when using these reagents.

Please visit our website www.biotium.com to download a comprehensive safety report.

GelRed™ and GelGreen™ passed the EPA regulated Title 22 test. Some facilities have approved the disposal of GelRed™ and GelGreen™ directly down the drain. However, because regulations vary, please contact your safety office for local disposal guidelines. Please review the GelRed/GelGreen safety report for more detailed information.

GelRed and GelGreen can be added to agarose during gel casting at a final concentration of 1X, or they can be used for post-electrophoresis gel staining at a final concentration of 3X in water. For detailed protocols for use, please download the GelRed™ Product Information Sheet or GelGreen™ Product Information Sheet.

Many customers use GelRed™ or GelGreen™ precast gels for convenience. However, because GelRed™ and GelGreen™ are high affinity dyes designed to be larger dyes to improve their safety, they can affect the migration of DNA in precast gels. Some samples, such as restriction digested DNA may migrate abnormally in GelRed™ or GelGreen™ precast gels.

Tip #1: Load less DNA

Smearing and smiling in GelRed™ or GelGreen™ precast gels most often caused by overloading of DNA. If you see band migration shifts or smearing and smiling, try reducing the amount of DNA loaded. The recommended loading amount for ladders and samples of known concentration is 50-200 ng/lane. For samples of unknown concentration, try loading one half or one third of the usual amount of DNA. This usually solves band migration problems.

Tip #2: Try the post-staining protocol

To avoid any interference the dye may have on DNA migration, we recommend using the post-staining protocol. If your application requires loading more than the recommended amount of DNA, use the post-staining protocol. While we recommend post-staining gels for 30 minutes, you may be able see bands in as little as five minutes, depending on how much DNA is present. Post-staining solutions can be reused. See the GelRed™ Product Information Sheet or GelGreen™ Product Information Sheet for detailed protocols.

Other tips to improve agarose gel resolution:

  • If you see DNA migration issues or smearing after post-staining with GelRed or GelGreen, then the problem is not caused by the nucleic acid dye. Avoid overfilling gel wells to prevent smearing of DNA down the surface of the gel.
  • Pour a lower percentage agarose gel. Higher molecular weight DNA separates better with a lower percentage gel.
  • Change the running buffer. TBE buffer has a higher buffering capacity than TAE buffer.

The dye may have precipitated out of solution.

  1. Heat GelRed™ solution to 45-50°C for two minutes and vortex to dissolve.
  2. Store dye at room temperature to avoid precipitation.

You can store precast GelRed™ gels for up to a week, and GelGreen™ gels for up to a month. We recommend storing gels at room temperature in the dark. We no longer recommend storing gels at 4°C, because this can lead to dye precipitation and poor performance.

GelRed™ and GelGreen™ can be added to agarose during gel casting at a final concentration of 1X, or used for post-electrophoresis gel staining at a final concentration of 3X in water. Dilute the 10,000X stock 10,000-fold for 1X precast gels (for example, 5 uL for a 50 mL gel), or 3,333-fold for a 3X post staining solution (15 uL for a 50 mL solution).

A 0.5 mL vial of GelRed™ or GelGreen™ is can be used to prepare 100 minigels (50 mL each) using the precast protocol, or for post-electrophoresis staining of 33 minigels in 50 mL staining solution per gel. Post-staining solution also can be re-used for staining two or more gels. For detailed protocols for use, please download the GelRed™ Product Information Sheet or GelGreen™ Product Information Sheet.

GelRed™ is more stable than GelGreen™. We do not recommend storing GelRed™ in molten agarose for more than a few days.

GelRed™ and GelGreen™ are stable dyes. You can use the dyes in room light. However, we recommend storage of the dyes in the dark between uses. We have had a customer report using GelRed™ with success after accidentally leaving it in ambient light for one month.

Yes. However, if the sensitivity decreases, use a fresh solution of the dyes.

No, but de-staining with water can be performed if background is high.

We have tested a variety of DNA ladders from different suppliers with GelRed™ and GelGreen™ with good results. Similarly, any common DNA loading buffers can be used. Band migration problems or smearing or smiling DNA bands are most commonly caused by overloading of ladder. If you are experiencing band migration problems with your ladder, please try reducing the amount of ladder you load. We recommend loading 50-200 ng ladder per lane for GelRed™ or GelGreen™ precast gels. See the FAQ for smearing or smiling DNA bands for more tips.

Biotium also offers 1 kb and 100 bp DNA ladders in TE buffer or ready-to-use format in loading buffer. Ladders are supplied with a separate tube of 6X DNA Loading Buffer to use with your samples.

No. We do not recommend that GelRed™/GelGreen™ gels be reused after electrophoresis because the staining intensity can be decreased with sequential electrophoresis.

Yes, but it may be necessary to add some more dye to the re-melted gel for the best signal.

Yes. We recommend Qiagen or Zymoclean™ gel extraction kits or phenol-chloroform extraction to remove the dye from DNA. Some users have reported performing PCR on DNA in the presence of GelRed™ with no purification step, for example by incubating GelRed™-stained gel slices in TE buffer to extract DNA by passive diffusion for use in PCR, or by using a few microliters of molten agarose from GelRed™-stained gel slices containing DNA for PCR.

GelRed™ and GelGreen™ can be used to stain both ssDNA and RNA, but GelRed is about 5 times more sensitive for single-stranded nucleic acids than GelGreen™. Titration assays using a fluorescence microplate reader showed that the fluorescence signal of GelRed™ bound to ssDNA and RNA is about half that of GelRed™ bound to dsDNA.

We don’t recommend adding GelRed™ or GelGreen™ directly to loading buffer, because this can result in inaccurate band migration. Biotium offers 6X GelRed™ Prestain Loading Buffers designed for this application, although we do not recommended them for applications where precise DNA band sizing is required. For the most accurate determination of DNA band sizes, we recommend using GelRed™ post-staining (see the GelRed™ protocol for details).

GelRed™ and GelGreen™ are ultra-sensitive dyes. Some users have reported being able to detect bands containing less than 0.1 ng DNA. However, the sensitivity of the staining will depend on the instrument capability and exposure settings.

GelRed™ has been shown to bind DNA exclusively by intercalation (http://link.springer.com/article/10.1007/s00249-014-0995-4). GelGreen™ most likely binds by a combination of intercalation and electrostatic interaction.

GelRed™ and GelGreen™ do not migrate through the gel as easily as ethidium bromide. It is not necessary to add additional dye to the running buffer, and the gel will be stained more homogenously than a gel stained with ethidium bromide.

GelRed™ has been validated for Southern blotting. We recommend using the post-staining protocol for blotting applications.

Yes, customers have reported using GelRed™ in glyoxal and formaldehyde agarose gels for precast staining of RNA.

Yes. Please use the post-staining procedure for DGGE and EMSA gels. For PFGE gels, the pre-cast or post-staining protocol may be used.

Yes, GelRed™ and GelGreen™ can be used for Comet assay.

Yes, GelRed™ is compatible with alkaline running buffer.

Customers have reported using GelRed™ in cesium gradients. To extract GelRed™ from DNA after cesium banding, we recommend adding SDS to a final concentration of 0.1% before butanol extraction. Alternatively, chloroform can be used instead of butanol for extraction.

Customers report good results using ZymoClean™ Gel DNA Recovery Kit from Zymo Research, GenElute™ Agarose Spin Column from Sigma, PureLink® Quick Gel Extraction kit from Life Technologies, illustra GFX PCR DNA and Gel Band Purification kit from GE Healthcare, High Pure PCR Product Purification Kit from Roche Applied Sciences, and GenJET gel extraction kit from Thermo Scientific.

Yes, use the post-staining protocol for polyacrylamide gels. For polyacrylamide gels containing 3.5-10% acrylamide, typical staining time is 30 minutes to 1 hour with gels of higher acrylamide content requiring longer staining time.

Biotium also offers PAGE GelRed™ and PAGE GelGreen™, which are non-toxic, non-mutagenic dyes specifically designed for staining DNA in polyacrylamide gels.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

PMA and PMAxx™ for viability PCR (10)

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Biotium offers the PMA-Lite™ LED Photolysis Device for light-induced cross-linking of PMAxx™ and PMA to dsDNA. The PMA-Lite™ is designed for samples in microcentrifuge tubes.

For users that have samples in a different format (for example, 96-well plates or filters), we will soon be releasing a new light box that will allow for greater flexibility of assay format.

Commercial halogen lamps (>600 W) for home use have been employed for photoactivating PMA in some publications, though results have not been consistent due to inevitable variation in the set-up configurations. If you decide to use a halogen lamp, we recommend that you lay tubes on a block of ice set 20 cm from the light source, on a rocking platform to ensure continuous mixing. Set the lamp so that the light source is pointing directly downward onto the samples (up to 45° downward slant is OK). Expose samples to light for 5-15 min.

The LEDs in PMA-Lite have brightness of 600-800 millicandela (mcd). There are three LEDs next to each tube (one bottom, two side) and the wavelength is 465-475 nm.

Both PMA and PMAxx™ are both offered as 20 mM solutions in water. We also offer PMA as a solid, and customers often ask whether it needs to be dissolved in DMSO. While in some publications users have prepared their PMA stock solution in DMSO, this is not necessary. Unlike the older viability dye EMA, which is not soluble in water, PMA and PMAxx™ are very water soluble.

We have compared PMA and PMAxx™ for their ability to differentiate between live and dead cells in viability PCR assays, and in all bacteria strains tested*, PMAxx™ had better activity. Therefore, while we haven’t tested every bacterial strain, we recommend PMAxx™ for bacterial viability PCR.

A recent publication by Randazzo et al. compared several viability PCR dyes in norovirus, and found that PMAxx™ worked the best for that organism.

We have also compared PMA and PMAxx™ in viability PCR assays with the yeast Saccharomyces cerevisiae, and found that PMA was equal to or better than PMAxx™. Therefore we recommend PMA for yeast and fungus viability PCR.

*E. coli, Salmonella enterica, Listeria monocytogenes, Bacillis subtilis, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Lactobacillus casei.

Many users have had success using PMA in viability PCR with environmental samples, but these samples can be more challenging than pure cultures. We’ve outlined some of the challenges below.

Water samples In order to perform viability PCR on water samples, some users have developed methods to concentrate the microorganisms onto a 0.45 um filter, and then treat the filter with PMA. An example protocol can be found in this publication: Ditommasso S., et al. (2015) Viability-qPCR for detecting Legionella: Comparison of two assays based on different amplicon lengths. Mol. Cell Probes. doi: 10.1016/j.mcp.2015.05.011. 

Opaque or complex samples Viability PCR with PMA has been successfully reported in various complex sample types, such as sewage or soil. No reports have been made yet with PMAxx™ in complex samples, but we would expect it to work as well or better than PMA. The main considerations for complex samples are dye concentration and light penetration. Typically a higher dye concentration (100 uM or more) is needed because some dye may bind to contaminants in the sample. Longer light treatment and more mixing during light treatment may be required, since it will be harder for the light to penetrate through the sample. An example protocol can be found in this publication: Guo F. and Zhang T. (2014) Detecting the nonviable and heat-tolerant bacteria in activated sludge by minimizing DNA from dead cells. Microb. Ecol. dio 10.1007/s00248-014-0389-2.

Viability PCR using PMA has been published in hundreds of publications, with dozens of different organisms, and many different types of samples. For more information, please see our PMA publication list or do a literature search for your application of interest.

PMA and PMAxx™ dyes are quite stable, and are expected to keep their activity after being left at ambient temperature, provided they were not exposed to light. We always suggest that you perform a small test experiment if you are concerned that a dye may have lost activity.

PMA and PMAxx™ dyes are very sensitive to light. Light exposure, even ambient room light, causes a chemical change in the dye molecule. Therefore we recommend that vials of PMA or PMAxx™ dyes only be opened in darkened rooms or very dim light. We always suggest that you perform a small test experiment if you are concerned that a dye may have lost activity.

In our standard viability PCR assay protocols we say that following PMA treatment, DNA should be purified from the cells before being used as a template for PCR. However there have been reports of the successful use of bacterial cell lysates in viability PCR following PMA treatment. The main consideration is to ensure that all of the dye is efficiently photolysed before preparing the lysate.  Unreacted PMA dye in the lysate could potentially inhibit the PCR reaction by binding to the DNA or polymerase. If you are considering trying this, we would suggest pelleting the cells after dye and light treatment to remove the excess free dye from the solution. In addition, control experiments should be performed to determine the ideal PMA and light treatment conditions that result in no PCR inhibition.

For some strains of bacteria, we sell complete viability PCR kits, which include strain-specific primer sets that have been validated at Biotium, as well as in publications in most cases. However any validated primer sets may be used to target your species of interest. We and others have found that amplicon length can affect your viability PCR results. Using longer amplicons generally results in better suppression of dead cell PCR products. For an example see this publication: Banihashimi A., et al. (2012) Long-amplicon propidium monoazide-PCR enumeration assay to detect viable Campylobacter and Salmonella. J. Appl. Miobiol. dio 10.1111/j.1365-2672.2012.05382.x.

While you can perform viability PCR with amplicons as short at 100 bp, we advise that you use longer amplicons for better results.

 

No, the PMA Enhancer should only be used when only gram- bacteria will be studied. The PMA Enhancer has a detrimental effect of gram+ bacteria.

Mix-n-Stain™ Antibody Labeling Kits (29)

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CF dyes are highly water soluble, small organic dyes designed by scientists at Biotium for labeling proteins and nucleic acids. With a series of over 20 colors (and growing), many of our CF dyes are brighter and more photostable than competing dyes. For more information please see the product flyers for individual CF dyes, the CF Dye Selection Guide, and our CF Dye FAQs.

See our CF™ Dye Quick Reference Table for a list of dyes and summary of their features. Our CF™ Dye Selection Guide has more detailed information on each CF™ dye, and ordering information for our various CF™ dye product lines.

See the Mix-n-Stain Kit Selection Guide, below:

Antibody formulationKit and protocol selectionNotes/Examples
Purified IgG containing:

• Sodium azide

• Less than 20 mM Tris

• Less than 10% glycerol
1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol
If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• More than 20 mM Tris

• More than 10% glycerol

• Glycine

• Less than 0.5 ug/uL IgG
1. Choose the kit size based on the amount of IgG you wish to label

2. Perform ultrafiltration using the spin vial provided in the kit

3. Use the standard labeling protocol
If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• Less than 4:1 BSA:IgG by weight

• Less than 4:1 gelatin:IgG by weight
1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol
For example, if you wish to label 5 ug IgG in 5 uL PBS contain­ing 0.1% BSA. The BSA:IgG ratio by weight is 5 ug BSA:5 ug IgG or 1:1. Select a 5-20 ug-sized kit and follow the standard protocol.
Purified IgG containing:

• More than 4:1 BSA:IgG by weight

• More than 4:1 gelatin:IgG by weight
1. Choose the kit size based on the total amount of protein (IgG + BSA/gelatin) in the volume of antibody solution you wish to label

2. Use the modified labeling protocol
For example, if you wish to label 5 ug IgG in 5 uL PBS containing 1% BSA. The BSA:IgG ratio by weight is 50 ug BSA: 5 ug IgG or 10/1. Select a 50-100 ug-sized kit based on 55 ug of total protein in the labeling reaction and follow the modified labeling protocol. If the total protein amount falls between two kit sizes, you may get better results with the larger kit size.
IgG in ascites fluid1. Determine the concentration of protein in the ascites fluid

2. Choose the kit size based on the total amount of protein in the volume of ascites fluid you wish to label

3. Use the modified labeling protocol
For example, if you wish to label 10 uL ascites fluid containing 70 ug total protein. Select a 50-100 ug-sized kit based on 70 ug of total protein, and follow the modified labeling protocol.
Less than 5 ug purified IgG1. Add stabilizer protein such as BSA to the antibody to bring the total amount of protein 5 ug

2. Select a 5-20 ug kit and use the standard labeling protocol
For example, if you wish to label 1 ug IgG sample. Select a 5-20 ug-sized kit. Before labeling, add 4 ug BSA to 1 ug IgG and follow the standard labeling protocol.
IgG in:

• Serum

• Hybridoma cell culture supernatant
Not compatible due to the low concentration of IgG compared to other proteins in these formats; purify IgG before labelingUse protein A/G or a commercially available IgG clean-up kit to purify IgG. Determine the concentration of IgG, then select the appropriate kit/protocol based on the amount of IgG you wish to label and the buffer formulation after purification.

There is no need to measure the dye amount or vary the reaction time as long as the amount of your antibody to be labeled falls within the range specified for each kit. With Mix-n-Stain labeling kits optimal labeling is ensured because of the proprietary dyes and reaction buffer.

The kits are optimized for labeling antibodies with a concentration between 0.5-1.0 mg/mL. If your antibody solution is too dilute, you can concentrate it by centrifugation using the ultra-filtration vial provided in the kit. If your antibody solution is too concentrated, you can dilute it with 1x PBS. Antibody concentrations outside the recommended range may result in either under or over labeling.

The dye is covalently attached to the side chains of amino acids located far away from the antigen-binding sites so that the affinity of the antibody is not affected. However, the exact attachment sites and the nature of chemical linkages are proprietary information.

This question relates to a key element of our invention. The unique formulations of our dyes and buffers and the labeling strategy have completely removed this concern, which normally has to be dealt with when using conventional antibody labeling methodology. The exact mechanism on how this problem is solved is proprietary information. For use on live cells we do recommend a quick purification using the provided ultrafiltration vial or staining at 4°C to prevent possible endocytosis of free dye.

Approximately 100%.

It takes 30 seconds or less to mix the components (antibody, dye and reaction buffer). After another 30 minutes of hands-free reaction time, you are done! You can use the antibody right away to stain your sample for microscopy, flow cytometry, Western analysis or other applications requiring fluorescently labeled antibodies.

The labeling reaction takes only 30 min. However, longer reaction time will not adversely affect the labeling.

The optimal reaction time is 30 minutes. However, shortening the reaction to 20 minutes still produces good results. If the reaction time is less than 30 min, you must combine the labeled antibody solution with the storage buffer provided before you use it to stain your sample.

Your labeled antibody is stable for at least six months if stored in the Mix-n-Stain storage buffer at 4°C . For longer-term storage, you can aliquot your antibody and store at -20°C.

Mix-n-Stain labeling results in covalent linkage of dye and antibody, so there will be no dye diffusion or transfer.

Direct immunofluorescence detection can be less sensitive than indirect detection. You may need to use a higher concentration of antibody or higher gain settings to achieve similar staining intensity compared to indirect immunofluorescence staining. In our internal testing, indirect immunofluorescence staining results in about 3-fold signal amplification compared to direct immunofluorescence staining.

Direct immunofluorescence staining eliminates the need for secondary antibody incubation and wash steps, and allows the use of multiple primary antibodies from the same species for multicolor detection, or staining of animal tissues with antibodies raised in the same species without secondary antibody cross-reactivity (e.g. mouse-on-mouse staining).

  1. Unlike Zenon, Mix-n-Stain labeling covalently attaches the dye to the antibody, eliminating dye transfer or diffusion between antibodies during multi-color staining.
  2. Mix-n-Stain conjugates are stable for at least 6 months in storage buffer, whereas Zenon complexes must be used within 30 minutes.
  3. Mix-n-Stain conjugates are less bulky because the dyes are directly linked to the antibody, unlike Zenon conjugates which use antibody fragments.
  4. No post-staining fixation is required with Mix-n-Stain.
  5. Unlike Zenon, Mix-n-Stain labeling is not species-specific.

Mix-n-Stain antibody labeling kits use novel CF dyes, which are brighter and more photostable than the dyes provided in Lightning Link kits. Mix-n-Stain kits are sized for labeling smaller amounts of antibody and are sold as single labelings, providing more flexibility compared to Lightning Link kits.

We have had customers report successful labeling of nanobodies (single domain antibodies) with Mix-n-Stain Antibody Labeling Kits. Mix-n-Stain kits are optimized for labeling whole IgG (150 kDa) with 4-6 dyes per antibody. Due to their smaller size (~15 kDa), single chain antibodies have fewer potential labeling sites. So to increase the degree of labeling for single chain antibodies, we recommend using a higher ratio of dye to protein. For example, the Mix-n-Stain 50-100 ug size kit could be used to label 25-30 ug of nanobody. Also, we do not recommend using the 10K MWCO ultrafiltration vial with proteins smaller than 30 kDa. If ultrafiltration is required prior to Mix-n-Stain labeling, a 3K MWCO ultrafiltration vial (catalog no. 22018) should be used instead.

Mix-n-Stain kits are optimized for labeling IgG antibodies, but can be used to label other proteins. Customers have reported successful labeling of nanobodies and single chain antibodies. Mix-n-Stain kits labeling conditions may cause denaturation of IgM antibodies. There are also published reports of Mix-n-Stain labeling of enzymes and lectins. Note that any conjugation method, including Mix-n-Stain, may affect the biological activity of proteins. Also, some free unreactive dye may remain after Mix-n-Stain labeling, which could interfere with live cell staining or trafficking studies using fluorescently labeled proteins. The ultrafiltration vial provided in the kit can be used to remove free dye after labeling if necessary.

Also see our CF Dye SE Protein Labeling Kits, which include everything you need to label and purify up to 3 mg protein. We also offer VivoBrite Antibody Labeling Kits for labeling antibodies or other proteins with near-infrared CF dyes for small animal in vivo imaging.

No. The Mix-n-Stain kits are optimized for 1 labeling. We do not recommend trying to split the kit to label more than one antibody or for more than one use.

PBS, HEPES, MES, MOPS or borate buffers are compatible. Buffers containing up to 20 mM Tris are also compatible. Tris at levels higher than 20 mM should be removed using the ultrafiltration vial provided in the kit. See Product Information sheet for more information.

Sodium azide, EDTA, small sugars, and <10% glycerol have no effect on the labeling. Higher levels of glycerol, or any level of DTT, 2-mercaptoethanol or free amino acids (such as glycine) should be removed using the ultrafiltration vial provided in the kit. See Product Information sheet for more information.

Labeling IgG free of BSA or gelatin stabilizer gives the best results. However, IgG containing BSA or gelatin can be labeled with good results using a larger sized kit and our modified Mix-n-Stain protocol. See the Mix-n-Stain Kit Selection Guide below to select the appropriate kit size and protocol.

Antibody formulation Kit and protocol selection Notes/Examples
Purified IgG containing:

• Sodium azide

• Less than 20 mM Tris

• Less than 10% glycerol

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• More than 20 mM Tris

• More than 10% glycerol

• Glycine

• Less than 0.5 ug/uL IgG

1. Choose the kit size based on the amount of IgG you wish to label

2. Perform ultrafiltration using the spin vial provided in the kit

3. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• Less than 4:1 BSA:IgG by weight

• Less than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS contain­ing 0.1% BSA. The BSA:IgG ratio by weight is 5 ug BSA:5 ug IgG or 1:1. Select a 5-20 ug-sized kit and follow the standard protocol.
Purified IgG containing:

• More than 4:1 BSA:IgG by weight

• More than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the total amount of protein (IgG + BSA/gelatin) in the volume of antibody solution you wish to label

2. Use the modified labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS containing 1% BSA. The BSA:IgG ratio by weight is 50 ug BSA: 5 ug IgG or 10/1. Select a 50-100 ug-sized kit based on 55 ug of total protein in the labeling reaction and follow the modified labeling protocol. If the total protein amount falls between two kit sizes, you may get better results with the larger kit size.
IgG in ascites fluid 1. Determine the concentration of protein in the ascites fluid

2. Choose the kit size based on the total amount of protein in the volume of ascites fluid you wish to label

3. Use the modified labeling protocol

For example, if you wish to label 10 uL ascites fluid containing 70 ug total protein. Select a 50-100 ug-sized kit based on 70 ug of total protein, and follow the modified labeling protocol.
Less than 5 ug purified IgG 1. Add stabilizer protein such as BSA to the antibody to bring the total amount of protein 5 ug

2. Select a 5-20 ug kit and use the standard labeling protocol

For example, if you wish to label 1 ug IgG sample. Select a 5-20 ug-sized kit. Before labeling, add 4 ug BSA to 1 ug IgG and follow the standard labeling protocol.
IgG in:

• Serum

• Hybridoma cell culture supernatant

Not compatible due to the low concentration of IgG compared to other proteins in these formats; purify IgG before labeling Use protein A/G or a commercially available IgG clean-up kit to purify IgG. Determine the concentration of IgG, then select the appropriate kit/protocol based on the amount of IgG you wish to label and the buffer formulation after purification.

IgG in ascites fluid can be labeled with good results using a larger sized kit and our modified Mix-n-Stain protocol. See the Mix-n-Stain Kit Selection Guide below to select the appropriate kit size and protocol. Mix-n-Stain labeling does not work well for antibodies in serum or hybridoma cell culture supernatant. We recommend purifying IgG before labeling. See Guide below for more information.

Antibody formulation Kit and protocol selection Notes/Examples
Purified IgG containing:

• Sodium azide

• Less than 20 mM Tris

• Less than 10% glycerol

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• More than 20 mM Tris

• More than 10% glycerol

• Glycine

• Less than 0.5 ug/uL IgG

1. Choose the kit size based on the amount of IgG you wish to label

2. Perform ultrafiltration using the spin vial provided in the kit

3. Use the standard labeling protocol

If the amount of antibody you wish to label falls between two kits sizes, we recommend using the smaller kit size. For example, if you wish to label 20 ug IgG, choose the 5-20 ug-sized kit.
Purified IgG containing:

• Less than 4:1 BSA:IgG by weight

• Less than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the amount of IgG you wish to label

2. Use the standard labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS contain­ing 0.1% BSA. The BSA:IgG ratio by weight is 5 ug BSA:5 ug IgG or 1:1. Select a 5-20 ug-sized kit and follow the standard protocol.
Purified IgG containing:

• More than 4:1 BSA:IgG by weight

• More than 4:1 gelatin:IgG by weight

1. Choose the kit size based on the total amount of protein (IgG + BSA/gelatin) in the volume of antibody solution you wish to label

2. Use the modified labeling protocol

For example, if you wish to label 5 ug IgG in 5 uL PBS containing 1% BSA. The BSA:IgG ratio by weight is 50 ug BSA: 5 ug IgG or 10/1. Select a 50-100 ug-sized kit based on 55 ug of total protein in the labeling reaction and follow the modified labeling protocol. If the total protein amount falls between two kit sizes, you may get better results with the larger kit size.
IgG in ascites fluid 1. Determine the concentration of protein in the ascites fluid

2. Choose the kit size based on the total amount of protein in the volume of ascites fluid you wish to label

3. Use the modified labeling protocol

For example, if you wish to label 10 uL ascites fluid containing 70 ug total protein. Select a 50-100 ug-sized kit based on 70 ug of total protein, and follow the modified labeling protocol.
Less than 5 ug purified IgG 1. Add stabilizer protein such as BSA to the antibody to bring the total amount of protein 5 ug

2. Select a 5-20 ug kit and use the standard labeling protocol

For example, if you wish to label 1 ug IgG sample. Select a 5-20 ug-sized kit. Before labeling, add 4 ug BSA to 1 ug IgG and follow the standard labeling protocol.
IgG in:

• Serum

• Hybridoma cell culture supernatant

Not compatible due to the low concentration of IgG compared to other proteins in these formats; purify IgG before labeling Use protein A/G or a commercially available IgG clean-up kit to purify IgG. Determine the concentration of IgG, then select the appropriate kit/protocol based on the amount of IgG you wish to label and the buffer formulation after purification.

Mix-n-Stain™ kits contain a very small quantity of dye or biotin, which usually is not visible in dry form. Once you add your antibody solution to the vial and mix, the solution will become brightly colored. Biotin, CF™350, CF™405M, and CF™405S Mix-n-Stain™ compounds yield colorless or very pale solutions, but rest assured that the vial does contain the dye or biotin.

  1. Check with the antibody manufacturer to confirm that the antibody formulation and concentration are compatible with the kit labeling protocol you selected.
  2. You should confirm that your primary antibody is sensitive and specific for your application using indirect labeling before attempting direct labeling. You may need to use a higher concentration of primary antibody to achieve similar signal intensity with direct labeling as with indirect labeling.
  3. Covalent labeling may affect the reactivity of certain antibodies. You can confirm that the labeled antibody is still reactive by performing indirect immunofluorescence labeling with your Mix-n-Stain labeled primary followed by a fluorescently-labeled secondary antibody.
  4. You can confirm labeling of your antibody by performing denaturing SDS-PAGE on a small amount (0.1-0.5 ug) of labeled antibody, then imaging the gel fluorescence at the appropriate excitation/emission wavelengths of the CF dye you used. You should be able to detect fluorescent bands representing IgG heavy and light chains at ~55 kDa and ~25 kDa.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

EvaGreen® dye and Master Mixes (8)

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EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix. However, there are several differences between the dyes.

Lower background fluorescence: EvaGreen® dye has less background than SYBR® Green I due to its novel “release-on-demand” DNA-binding mechanism.

Brighter and non-inhibitory:  EvaGreen® is less inhibitory in the PCR reaction than SYBR® Green, permitting the use of saturating dye concentration for maximal signal and better high-resolution DNA melt analysis.

Safer and more environmentally friendly: EvaGreen® dye is the first and only PCR dye to date designed to be environmentally safe.

Dye stability: EvaGreen® dye is very stable both during storage and under PCR conditions. SYBR® Green I, on the other hand, is known to degrade following multiple freeze-thaw cycles and under PCR conditions. Moreover, decomposed SYBR® Green I is reported to be even more inhibitory to PCR.

Digital PCR: EvaGreen® dye is currently the only qPCR dye to be used in droplet digital PCR (ddPCR).

Biotium offers three different EvaGreen® dye-based qPCR master mixes:

  • Forget-Me-Not™ qPCR Master Mix
  • Fast EvaGreen® qPCR Master Mix
  • Fast Plus EvaGreen® qPCR Master Mix

All three master mixes contain our high-quality EvaGreen® DNA-binding dye and our fast-activating chemically-modified hotstart enzyme, Cheetah™ Taq.

Forget-Me-Not™ qPCR Master Mix is our newest master mix formulation. For most purposes this is the master mix that we would recommend, as it has many benefits: best sensitivity and signal, blue tracking dyes in the master mix and template to reduce pipetting errors, and low cost. Fast EvaGreen® qPCR Master Mix is a sensitive, general use master mix that does not contain blue tracking dyes. Fast Plus EvaGreen® qPCR Master Mix has a slightly different formulation than the regular Fast EvaGreen® qPCR Master Mix, and was designed for more challenging samples.

Both of these qPCR master mixes contain Cheetah™ Taq (our chemically modified hotstart DNA polymerase), buffer and dNTPs.

The Fast EvaGreen® Master Mixes also contain the DNA-binding dye EvaGreen®, and are used for fluorescent dye-based qPCR. The only thing the user needs to supply are primers and template.

The Fast Probe Master Mix  is formulated for conducting real-time PCR using an oligo-based probe, such as a TaqMan™ probe, an MGB probe or an AllGlo™ probe. The user needs to supply primers, probe and template.

Forget-Me-Not™ qPCR Master Mix is a hot-start EvaGreen® dye-based master mix. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. This master mix also contains a light blue tracking dye, and comes with a dark blue template dye, for two-color tracking to reduce pipetting errors. The user only needs to supply primers and template.

Forget-Me-Not™ Universal Probe Master Mix is formulated for fluorescent probe-based PCR applications. It uses Biotium’s proprietary chemically modified hot-start DNA Polymerase, Cheetah™ Taq. The user needs to supply some kind of fluorescent probe (such as TaqMan®, dual-labeled BHQ®, or Molecular Beacon) in addition to primers and template.

Our qPCR Master Mixes are all offered with optional ROX reference dye, which is used by some qPCR instruments to normalize the PCR fluorescence. We also include instructions for using no ROX, low ROX, or high ROX, depending on the instrument you are using. Based on instrument manufacturers’ recommendations, we recommend the following:

No ROX

BioRad: iCycler™, MyiQ™, MiQ™2, iQ™5, CFX-96 Touch™, CFX-384 Touch™, Chromo4™, MiniOpticon™

Qiagen: Rotor-Gene® Q, Rotor-Gene® 3000, Rotor-Gene® 6000

Eppendorf: Mastercycler® Realplex

Illumina: Eco™ RealTime PCR System

Cepheid: SmartCycler®

Roche: LightCycler® 480, LightCycler® 2.0

Low ROX

ABI: 7500, 7500 Fast, ViiA 7™, QuantStudio™

Stratagene: MX4000P, MX3000P, MX3005P

High ROX

ABI: 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne®, StepOne Plus®

EvaGreen® dye is spectrally similar to FAM or SYBR® Green I, which means no change in optical settings is required for using an EvaGreen®-based master mix compared to SYBR® Green-based master mixes. You can read your signal in the green fluorescence channel (FAM, SYBR® Green, or EvaGreen®) of your instrument.

For most qPCR applications, we recommend using EvaGreen® Dye, 20X  in water (31000), or one of our 2X EvaGreen® qPCR Master Mixes. EvaGreen® Dye, 2000X  in DMSO (31019) is offered for specialized applications requiring a concentrated dye solution.

If necessary, EvaGreen® Dye, 2000X  in DMSO can be diluted in water or master mix for qPCR for a final concentration of 1X in the PCR reaction.

DNA Quantitation Kits (16)

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AccuGreen™ kits are designed for use with the Qubit® fluorometer. All of the other kits are designed for use with 96-well fluorescence plate readers. Our quantitation kits vary in the concentration ranges of dsDNA that they are able to detect. Some of the kits also have different fluorescence excitation/emission. Please see the AccuBlue, AccuClear and AccuGreen Technology Highlights to choose the kit that best fits your application.

The AccuClear™ and AccuBlue™ DNA Quantitation kits are designed for use with fluorescence 96-well plate readers. AccuClear™, AccuBlue™ NextGen and AccuBlue™ High Sensitivity kits require an instrument equipped to read green fluorescence emission (similar to FITC). AccuBlue™ Broad Range requires an instrument equipped to read blue fluorescence emission (Ex/Em 350/460 nm).

These assays also can be used with fluorometers such as the Qubit® (Thermo Scientific) and QuantiFluor™-P (Promega). However, due to different linear ranges of the assays, not all of these assays are compatible with the pre-programmed DNA quantitation programs on these instruments. For users who own a Qubit® fluorometer, we recommend using our AccuGreen™ kit, which is designed for use on that instrument.

The AccuGreen™ High Sensitivity DNA Quantitation kit is designed for use on the Qubit® fluorometer. It can be used in the preprogrammed Qubit® dsDNA High Sensitivity assay, and is a direct replacement for the Qubit® dsDNA HS Assay kit.

For detection of picogram levels of DNA on a microplate reader, we recommend our AccuBlue™ NextGen dsDNA Quantitation Kit (detection range 2.5 pg-3 ng dsDNA) which has higher accuracy and sensitivity than PicoGreen® in a 96-well assay format.

Also see our AccuClear™ Ultra High Sensitivity dsDNA Quantation Kit, which is a great all-purpose choice: it has high sensitivity with a broad linear detection range (30 pg-250 ng dsDNA).

If you own a Qubit® fluorometer, we recommend our AccuGreen™ High Sensitivity dsDNA Quantitation kit, which is designed for use with the Qubit® fluorometer. It is a direct replacement for the Qubit® dsDNA HS Assay kit.

Note that the lower limits of kit sensitivity may depend on the detection instrument used.

A Quantitation Kit contains the quantitation solution components and pre-diluted calf thymus DNA standards. The Quantitation Solution contains the quantitation solution components, and does not include DNA standards.

No. It is recommended to use a DNA standard that most closely resembles your samples. For example, for bacterial DNA quantitation, you may prefer to use lambda DNA as a standard.

Yes, for some of the kits. You can purchase a set of AccuBlue™ High Sensitivity standards (cat. no. 31006C) or AccuBlue™ Broad Range standards (cat. no. 31007C) separately.

The dyes are selective for dsDNA; however there is some binding to RNA and ssDNA. Therefore, for the most accurate quantitation, it is recommended to use as clean a dsDNA preparation as possible. Please see the product information sheet for detailed information on RNA and ssDNA binding.

To minimize cost, the trial size kits come with a single DNA standard solution and instructions for preparing a set of standards by dilution. Most of the full size kits are provided with prepared standard sets. The exception is the AccuBlue™ NextGen kit, which is supplied with a single standard of 10 ng/uL, with instructions on diluting it to the lower concentrations used in the assay. We found that lower concentrations of DNA were not stable enough for long-term storage.

The AccuBlue™ High Sensitivity dye is membrane impermeable to live cells, while PicoGreen® has been shown to readily enter cells. We have not performed such testing with the dyes from our other DNA quantitation kits.

It is highly recommended to minimize cross-over of signals between wells. Depending on your instrument, you can use all black plates or plates with black-walls and clear bottoms.

Some of our DNA quantitation kits contain an enhancer in addition to the DNA quantitation dye. These kits have been optimized with the enhancer to maintain sensitivity and linearity. Omitting the enhancer may affect your results.

Yes. It is recommended to use as clean a dsDNA preparation as possible, but the kits can tolerate some common contaminants. Each kit varies in how it affected by various contaminants. Please see the product information sheets for each kit more information.

The assays can be scaled down for different well sizes. Scale the volumes of all reagents and DNA proportionally. The ratios of DNA, dye, and enhancer (if applicable) should remain the same as in the standard 96-well protocol.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

NucView™ Caspase 3 Enzyme Substrates (13)

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The substrate is very stable. Some users have reported performing time course assays with NucView™ 488 Caspase-3 Substrate for 4-5 days.

NucView™ Caspase-3 Substrates can be added to the cells at the start of the experiment or at the end. A major advantage of NucView™ Caspase-3 Substrates compared to other apoptosis assays is that it can be used to monitor capase-3 activity in real time.

Yes. NucView™ Caspase-3 Substrates are compatible with flow cytometers and other instruments with the appropriate excitation and emission settings.

Yes. We recommend a gentle fix in 2-4% paraformaldehyde for 10-15 minutes. Over-fixing can cause the signal to decrease. NucView™ 488 staining can withstand permeabilization with 0.1% Triton X-100. Methanol fixation is not recommended.

NucView™ Caspase-3 Substrates have not been validated by Biotium for live tissue staining. However, we have had feedback from a customer who successfully used NucView™ 488 Caspase-3 Substrate for live zebrafish embryo staining. NucView™ Caspase-3 Substrates cannot be used in fixed cells or tissues.

As with all fluorescence based probes, bleaching will occur over time. How long you can view NucView™ staining under the microscope depends on several factors including the initial signal strength and the intensity of the excitation source.

DEVD-CHO is a reversible inhibitor and may not sufficiently block caspase-3 activity. Adding an irreversible inhibitor like DEVD-FMK at the beginning of the experiment (before or at the time of apoptosis induction) may more effectively inhibit caspase activity.

Like other caspase-3 substrates, NucView™ 488 Caspase-3 Substrate is based on a DEVD sequence that also can be cleaved by caspase-7.

We currently offer NucView™ in three dye colors: green (our original NucView™ 488), blue (NucView™ 405), and orange (NucView™ 530). Other colors of NucView™ are in development.

Our NucView™ substrates detect cleavage by caspases 3 and 7. We do not currently have NucView™ substrates for other caspases.

The tables below list primary cells types and immortalized cell lines reported to work with NucView™ 488 in the published scientific literature. Click here for a PDF version of the tables along with the references.

Primary cell typeSpecies
Alveolar epithelial cellsMouse
Cortical neuronsRat
Dendritic cellsMouse
Embryonic fibroblast (MEF)Mouse
Embryo tailbudChicken
Gingival fibroblastsHuman
GliaRat
HemocytesSilkworm(Bombyx mori)
HepatocytesRat
Hippocampal neuronsRat
Idiopathic pulmonary fibrosis fibroblastsHuman
Immature B cellsMouse
Kidney epithelial cellsMouse
Lung microvascular endothelial cellsHuman
MacrophagesMouse
Mammary epithelial cells (3-D cultures)Mouse
NeutrophilsHuman
SVZ neural progenitor cellsRat
OligodendrocytesMouse
OocytesBovine, mouse
Pancreatic acinar cellsMouse
Pancreatic beta cellsRat
Pancreatic islet cellsMouse
Peritoneal macrophagesMouse
Pollen tubesField poppy(Papaver rhoeas)
Retinal pigmented epithelial cellsHuman, mouse
Skin fibroblastsSand cat(Felis margarita)
Stem cellsHuman
ThymocytesMouse
Umbilical vein endothelial cellsHuman
Vascular endothelial cellsRat

 

Immortalized cell lineSpeciesCell type
293-HHumanEmbryonic kidney
293-THumanEmbryonic kidney
4T1MouseMammary tumor
67NRMouseMammary carcinoma
A172HumanGlioma
A204HumanSarcoma
B16F10MouseMelanoma
BeWoHumanTrophoblast
CCL-134HumanIPF pulmonary fibroblast
CCL-190HumanPulmonary fibroblast
CCRF-CEMHumanLeukemia
FU-UR-1HumanRenal cell carcinoma
GE11MouseEpithelial
H9c2RatCardiac myoblast
HaCaTHumanKeratinocyte
HCLEHumanCorneal epithelial
HeLaHumanCervical cancer
HepT1HumanHepatoblastoma
HMECHumanMicrovascular endothelial
HL-60HumanLeukemia
HOSHumanOsteosarcoma
HT-1080HumanBreast fibrosarcoma
HUH6HumanHepatoblastoma
JurkatHumanT-lymphocyte
JYHumanLymphoblastic leukemia
K562HumanMyelogenous leukaemia
LLC-PK1PigKidney epithelial
MCF-7HumanBreast adenocarcinoma
MCF-10AHumanBreast adenocarcinoma
MDA-MB-231HumanBreast adenocarcinoma
MDA-MB-468HumanBreast cancer
MDCKCanineKidney epithelial
MES-SAHumanUterine sarcoma
MES-SA/DXHumanUterine sarcoma
MG-63HumanOsteosarcoma
Min 6MousePancreatic insulinoma
MOLT-3HumanLeukemia
N19MouseOligodendrocyte
NRKRatKidney epithelial
NRK-52ERatKidney epithelial
PC-3HumanProstate cancer
PC12RatPheochromocytoma
RDHumanRhabdomyosarcoma
RINm5FRatInsulinoma
Saos-2HumanOsteosarcoma
SKBR3HumanBreast cancer
SKLMS1HumanLeiomyosarcoma
STHdhMouseStriatal cells
SW684HumanFibrosarcoma
SW872HumanLiposarcoma
THP-1HumanMonocyte
TK6HumanSplenic lymphoblast
U2OSHumanOsteosarcoma
U251HumanGlioblastoma
U373 MGHumanGlioblastoma
U937HumanLymphoma
WEHI 7.2MouseLymphoid

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Apoptosis, Necrosis, and Cell Viability Kits (13)

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MTT and XTT are colorimetric based assays, while resazurin can be measured using colorimetric or fluorescence detection. MTT is not a soluble product, so the cells must be lysed to solubilize the formazan salt before absorbance can be measured. XTT and resazurin do not require cell lysis, allowing kinetic monitoring of the same samples at different timepoints.

Check to make sure your cell densities are in the linear range of the cell viability assay. Too few cells may fall below the limit of detection, and too many cells may saturate the OD. Perform an initial experiment to check the best linear range for your cell type and experimental set-up. You may need to vary the cell density assay incubation time. The kit protocols provide general guidelines and may need to be optimized for your experimental system.

Resazurin is reduced by cells to the fluorescent product resorufin. Resorufin can be reduced further to a non-fluorescent compound. Therefore the densest wells may have lowest fluorescence due to over-reduction of the substrate. Please see the product information sheet for more details. The kit protocol provides general guidelines and may need to be optimized empirically for your experimental system. You may need to vary cell density or assay incubation time to ensure that your samples fall in the linear range of the kit.

AlamarBlue® contains resazurin and additional compounds to prevent the over-reduction of resazurin to a non-fluorescent product. These additives also slow the rate of generation of the fluorescent product. Consequently, the alamarBlue® assay requires longer incubation times compared to resazurin.

No. Fixation, freezing, sectioning, or dissociation of tissues can affect the PS on the outer leaflet and compromise membrane integrity. Both Annexin V and Ethidium Homodimer III rely upon the presence of intact membranes in healthy cells to accurately distinguish healthy cells from apoptotic or necrotic cells. To detect apoptosis in fixed cells and tissues we recommend our TUNEL kits. Currently we are not aware of a fluorescent probe that specifically detects necrotic cells in fixed tissues. Necrotic cells in tissue sections are identified based on morphological criteria.

The apoptosis, necrosis and cell viability assays are designed to stain dissociated cells in culture and have not been validated for organ culture. Annexin V staining of early chicken and mammalian embryos in culture has been reported in the scientific literature. For staining of living tissues, the specimen would need to be thin enough to allow exposure of the cells to the 36 kDa Annexin V protein. Also, damage to cell membranes from dissection or sectioning of tissues could result in high background staining.

Yes, cells can be fixed with formaldehyde after staining. Because Annexin V staining is dependent on calcium, all buffers used for washing and fixation should contain 1.25 mM CaCl2. Fixation may increase the background signal from Ethidium Homodimer III. Wash the cells several times to remove unbound Ethidium Homodimer III before fixation.

Annexin V is a 36 kDa protein that binds to the phospholipid phosphatidylserine. Therefore Annexin V binding is not species-specific.

The Annexin V protein that we use is a recombinant protein made in E. coli.

The binding buffer is an isotonic buffer containing calcium, which is essential for the binding of Annexin V to phosphatidylserine.

No, the concentration of Annexin V and Ethidium Homodimer III should be kept constant regardless of cell number. However, the staining concentrations can be increased or decreased if necessary to optimize staining.

You may wish to process floating and detached cells separately – collect the washes and spin them down and then stain those cells using the protocol for suspension cells. Alternatively you may collect floating cells, detach the adherent cells with trypsin (without EDTA), pool the cells together, and use the suspension cell protocol for staining.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Luciferase Assays (14)

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No. Biotium’s Renilla and Firefly Assay Kits are based on flash luminescence. Samples should be treated and read individually. Alternatively, you may purchase the Steady-Glo Firefly Assay Kit for HTS. We currently do not have a Steady-Glo for Renilla Assays.

The Passive Lysis Buffer (Renilla) is compatible with the Firefly Assay, but the Firefly Lysis Buffer is not compatible with the Renilla Assay.

Luminescence values are relative and depend on the experimental system. For example, luminometers from two different manufacturers each may have 6-log capability, but one may measure a range of 1 to 1,000,000 relative light units (RLU) while the other measures 0.01 to 10,000 RLU. Both instruments can measure a dynamic range of 6-log units, but they read out different arbitrary units.

Biotium’s Renilla and Firefly Assays are flash luminescence assays. The approximate half-lives are 1-2 minutes for Renilla and 10 minutes for Firefly. The Steady-Glo Firefly Assay has a half-life of about 3-5 hrs. However, the signal is about 1/10 of the flash luminescence assays.

No. The buffers are based on different proprietary formulations.

Renilla is often used as an internal control for transfection efficiency while the Firefly luciferase vector is used for reporter activity. They also can be reversed, with Renilla as the reporter and Firefly as the internal control.

Yes, please check the website for catalog numbers and pricing.

Assay sensitivity is dependent on many factors including the promoter strength of each vector, the transfection efficiency of each vector, and the cell type used. In general, Firefly luciferase is more widely used because it has a longer half-life and and better stability than Renilla luciferase.

Some of Biotium’s luciferase assay kits can be used as alternatives to Promega kits. The comparable products are listed below. Click on a link for product information.

Promega assayPromega catalog no.Biotium alternativeBiotium catalog no.Assay typeNotes
Steady-Glo® Assay SystemE2510, E2520, E2550Steady-Luc™ Firefly HTS Assay Kit30028Glow-type
Signal half-life 3h
Signal half-life ~3 hours. Luciferase substrate is added to assay buffer as needed, so signal doesn't drop during kit storage.
Steady-Luc™ Firefly HTS Assay Kit (Lyophilized)30028LGlow-type
Signal half-life 3h
Signal half-life ~3 hours. Lyophilized format for convenient shipping and storage.
Firefly Luciferase Assay SystemE1483, E1500, E1501, E4030, E4530, E4550Firefly Luciferase Assay Kit 2.030085Flash-typeLuciferase substrate is added to assay buffer as needed, so signal doesn't drop during kit storage.
Firefly Luciferase Assay Kit (Lyophilized)30075Flash-typeLyophilized format for convenient shipping and storage.
Renilla Luciferase Assay SystemE2810, E2820Renilla Luciferase Assay Kit 2.030082Flash-typeFeatures Biotium's water-soluble Aquaphile™ Coelenterazine
Dual-Luciferase® Assay System
(Firefly & Renilla)
E1910, E1960, E1980Firefly & Renilla Luciferase Single Tube Assay Kit30081Flash-typeSequentially measure firefly and Renilla luciferase activities in the same sample in one tube.
CellTiter-Glo® Luminescent Cell Viability AssayG7570, G7571, G7572, G7573ATP-Glo™ Cell Viability Assay30020Flash-typeCell Titer Glo® is a glow type assay (5h half-life); ATP-Glo™ is a flash type assay (signal stable for 1 minute)

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

CF™ Dyes (16)

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See our CF™ Dye Quick Reference Table for a list of dyes and summary of their features. Our CF™ Dye Selection Guide has more detailed information on each CF™ dye, and ordering information for our various CF™ dye product lines.

CF initially was an abbreviation for Cyanine-based Fluorescent dyes. These were the first patented CF dyes based on cyanine dye structures. Since then, our CF dye patent portfolio has expanded to include four different fluorescent dye core structures that cover the fluorescence spectrum from UV to NIR.

The exact chemical structures of CF dyes are currently confidential but will be fully disclosed at a later stage when pending patents become granted. In general terms, the structure of a CF dye may be divided into two parts: a) dye core structure (i.e. the aromatic ring skeleton that defines the dye’s color or absorption/emission wavelengths), and b) core structure-modifying elements. At present, CF dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes. Blue fluorescent CF dyes are based on coumarin or pyrene dye core structure, while green to near-IR CF dyes are based on either cyanine or rhodamine dye core structures. Core structure-modifying elements refer to various chemical attachments to the core structure and are a key aspect of the CF dye invention that makes CF dyes superior to other commercial dyes.

There is no simple answer to this question as the quantum yield of a fluorescent dye can vary widely, depending on the dye’s micro-environment. For example, the quantum yield of a dye attached to a protein may be very different from the quantum yield of the free dye. For dyes attached to a protein, the quantum yield is highly dependent on how many molecules of the dye are attached to the protein (i.e. degree of protein labeling). In general, a higher degree of protein labeling leads to a lower dye quantum yield due to fluorescence quenching via dye-to-dye interaction. For this reason, as the degree of labeling increases, fluorescence intensity of the labeled protein will eventually reach a maximum and start to decline thereafter. In fact, one of the best ways to compare the relative quantum yields of different dyes is to plot the total fluorescence of the labeled proteins as a function of degree of labeling by the dyes as we have done with CF dyes and other commercial dyes. CF dyes generally give higher slopes than other commercial dyes in the plots, suggesting less quantum yield decline with increasing degree of protein labeling.

We have not performed this measurement for CF dyes. However, some of our customers have obtained these values. We will compile the data as they become publicly available in the future.

There are usually three aspects to dye stability: 1) chemical stability of the dye core structure; 2) stability of the reactive group; and 3) photostability of the dye.

 
Chemical stability of the dye core structure:
This refers to resistance of the dye core structure to decomposition caused by factors other than photo-bleaching. These factors may include temperature, pH and incompatibility with other chemicals in the medium. This type of stability information is most useful for estimating the shelf-life of the dye that is already covalently attached to another molecule (e.g., an antibody), or for assessing the chemical compatibility of the dye in certain applications. CF dyes bear the core structures of coumarin, pyrene, rhodamine or cyanine dyes, all of which are known to have excellent chemical stability. In general, CF dyes are far more stable than the antibodies they label. Thus, if a CF-labeled antibody loses activity during storage, the problem is not likely to be caused by the dye. CF dyes are also stable enough for labeled nucleic acids to be used in PCR or nucleic acid hybridization, where high temperature is involved.
 
Stability of the reactive group:
Reactive CF dyes comprise a reactive group used in bioconjugation. Among the various reactive groups, only amine-reactive succinimidyl ester (SE) and thiol-reactive maleimide groups are unstable because the small amount of moisture trapped in or leaked into the packaging vials can cause hydrolysis of the reactive groups over time. The SE group, in particular, is susceptible to degradation. Thus, in order to slow degradation, CF dyes comprising these reactive groups must be stored at -20°C under anhydrous conditions. Furthermore, stock solutions of the dyes must be made using dry solvents, such as anhydrous DMSO. One advantage of CF dye SE products over other commercial dyes is their relatively high stability. Normally, an SE group can be derived from either an aliphatic or an aromatic carboxylic acid group, but an aliphatic carboxylic group tends to result in a more stable SE, offering higher resistance to hydrolysis and thus better labeling efficiency. All of the CF SE dyes have their SE groups derived from aliphatic carboxylic acid groups, unlike many of the Alexa Fluor SE dyes, which are prepared from aromatic carboxylic acid groups.
 
Photostability:
This refers to the dye’s ability to withstand photobleaching. For most dyes, photostability is not a major problem for routine handling under ambient light or for applications, such as flow cytometry and Western blotting, where the dyes are only briefly exposed to light. However, for microscopy, especially for confocal microscopy, where the dyes may be subject to intense illumination for an extended period of time, photobleaching can be a major concern. Similar to the photostability of other fluorescent dyes, both the dye core structure and the structure-modifying groups attached to it play a role in the photostability of CF dyes. CF dyes bear the core structure of rhodamine, cyanine, pyrene or coumarin dyes; among the four types of core structures, rhodamine core is the most photostable, followed by cyanine and then by pyrene and coumarin cores. The structure-modifying groups and the way they are attached to the dye cores are a key innovative aspect of CF dye technologies that contributes to the superior photostability of CF dyes over that of other commercial dyes. In general, rhodamine-based CF dyes, whose wavelengths range from visible to the near-IR region, offer the best photostability, making the dye ideal for microscopy applications.

CF dyes are chemically stable within the pH range of at least 2 –11. The fluorescence of most CF dyes is relatively insensitive to pH, except for that of CF405M, CF568, CF620R, and CF633. The fluorescence of these four CF dyes becomes weaker when pH drops below 4.5.

CF™ dyes can tolerate formaldehyde fixation. However, whether a CF™ dye-labeled probe is fixable will depend on the fixability of the probe itself. Proteins with free amine groups that bind other proteins generally are formaldehyde-fixable.

All three of these dyes can be excited by the 405 nm laser (or UV mercury lamp). They differ in their emission wavelengths. CF™405S has blue fluorescence emission at 431 nm, similar to AlexaFluor® 405, Cascade Blue®, and DyLight™ 405 (Thermo Scientific). CF™405M has longer wavelength blue fluorescence emission at 452 nm, similar to Horizon™ V450 (BD Biosciences) and Pacific Blue® (Thermo Scientific). CF™405L has orange fluorescence emission at 545 nm, similar to Pacific Orange® (Thermo Scientific). We recommend choosing the dye that best fits your instrument’s detection settings. See our CF™ Dye Quick Reference Table and CF™ Dye Selection Guide for more information on CF™ dyes.

Rhodamine-based CF dyes (designated R) generally have better photostability but weaker fluorescence than their cyanine-based equivalents (designated C). Therefore, rhodamine-based near-IR CF dyes are a better choice for microscopy, while cyanine-based CF dyes are more ideal for flow cytometry, Western blotting, and other applications where photobleaching is less of a concern. Another factor to consider is the size of the dyes. Some of the cyanine-based near-IR CF dyes are much larger than the rhodamine-based equivalents. For antibody labeling, either version of the CF dyes is suitable. However, for applications where the dye size may cause a steric problem, the smaller dye may be a better choice.

CF dyes are highly water soluble (>100 mg/mL). They are also very soluble in other polar solvents, such as DMSO, DMF, methanol and ethanol. However, CF dyes are poorly soluble or insoluble in non-polar solvents.

Most CF dyes carry 1-2 negative charges while a few cyanine-based near-IR CF dyes carry 3-4 negative charges. However, the more negatively charged CF dyes comprise unique structural features that shield the negative charges such that the biomolecules (such as antibodies) the dyes label do not lose specificity due to the excessive negative charges.

CF dyes are ideal for protein labeling because of their high water solubility, which reduces fluorescence quenching. They are also useful for labeling oligonucleotides that require multiple copies of a dye for maximal fluorescence, such as the preparation of FISH probes, where water soluble dyes can minimize fluorescence quenching. Finally, CF dyes make excellent polar tracers that can be used for visualizing the morphology or long-term tracing of neurons after microinjection. CF dyes and their conjugates are ideal for fluorescence microscopy applications, flow cytometry, Western blotting, and in vivo imaging (near-IR CF dyes).

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

CellBrite™ Cytoplasmic Membrane Stains (3)

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CellBrite cytoplasmic membrane stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain artificial lipid bilayers.

Lipophilic carbocyanine dyes have been used to stain neuronal cells in culture for several weeks, and in vivo for up to a year. The dyes do not appreciably affect cell viability, and do not readily transfer between cells with intact membranes, allowing cell migration and tracking studies in mixed populations. Stability of labeling may vary between cell types, depending on rates of membrane turnover or cell division.

Cells can be fixed with formaldehyde after labeling with CellBrite dyes. Lipophilic carbocyanine dyes like the CellBrite dyes have also been used to stain cells or tissues after formaldehyde fixation. Permeabilization of cells with detergents or solvents, or mounting medium containing glycerol may adversely affect staining. Permeabilization with digitonin (10 ug/mL to 1 mg/mL) has been reported to be compatible with lipophilic carbocyanine dye staining.

EverBrite™ Mounting Medium (1)

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EverBrite™ Mounting Medium (23001) and EverBrite™ Mounting Medium with DAPI (23002) both have a refractive index of 1.46.  EverBrite™ Hardset Mounting Medium (23003) and EverBrite™ Hardset Mounting Medium with DAPI (23004) both have an initial refractive index of 1.38, which increases to 1.42 after 24 hours of curing, and 1.46 after 4 days, after which it remains constant

Product shelf life, solubility, shipping and storage, and stability (7)

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Many of our solid compounds are packaged by lyophilization, in which case they usually do not appear as fluffy powders, but form a film or coating on the sides of the vial. Simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Bioscience kits
The guaranteed shelf life from date of receipt for bioscience kits is listed on the product information sheet. Some kits have an expiration date printed on the kit box label, this is the guaranteed shelf life date calculated from the day that the product shipped from our facility. Kits often are functional for significantly longer than the guaranteed shelf life. If you have an older kit in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the kit still works for your application before processing a large number of samples or precious samples.

Antibodies and other conjugates
The guaranteed shelf life from date of receipt for antibodies and conjugates is listed on the product information sheet. Antibodies and other conjugates often are functional for significantly longer than the guaranteed shelf life. If you have an older conjugate in storage that you wish to use, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

For lyophilized antibodies, we recommend reconstituting the antibody with glycerol and antimicrobial preservative like sodium azide for the longest shelf life (note that sodium azide is not compatible with HRP-conjugates).

Chemicals, dyes, and gel stains
Biotium guarantees the stability of chemicals, dyes, and gel stains for at least a year from the date you receive the product. However, the majority of these products are highly stable for many years, as long as they are stored as recommended. Storage conditions can be found on the product information sheet or product safety and data sheet, material safety data sheet, and on the product label. Fluorescent compounds should be protected from light for long term storage.

If you have a Biotium compound that has been in storage for longer than one year that you wish to use, we recommend performing a small scale positive control experiment to confirm that the compound still works for your application before processing a large number of samples or precious samples.

Expiration date based on date of manufacture (DOM)
If your institution requires you to document expiration date based on date of manufacture for reagents, please contact techsupport@biotium.com for assistance.

Chemical products with special stability considerations:

Esters

Ester compounds include the following:
• Succinimidyl esters (SE, also known as NHS esters), such as our amine-reactive dyes
• Acetoxymethyl esters (AM esters) such as our membrane-permeable ion indicator dyes
• Diacetate-modified dyes, like ViaFluor™ 405, CFDA, and CFDA-SE cell viability/cell proliferation dyes

Ester dyes are stable in solid form as long as they are protected from light and moisture. Esters are not stable in aqueous solution. Concentrated stock solutions should be prepared in anhydrous DMSO (see Biotium catalog no. 90082). Stock solutions in anhydrous DMSO can be stored desiccated at -20°C for one month or longer. Esters should be diluted in aqueous solution immediately before use. Succinimidyl esters (SE) should be dissolved in a solution that is free of amine-containing compounds like Tris, glycine, or protein, which will react with the SE functional group. AM esters and diacetate compounds should be dissolved in a solution that is free of serum, because serum could contain esterases that would hydrolyze the compound.

A note on CF™ dye succinimidyl ester stability
Succinimidyl esters are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Heavily sulfonated dyes, such as the Alexa Fluor® dyes, DyLight® dyes and IRDyes® are particularly hygroscopic, worsening the hydrolysis problem. For example, the percent of active Alexa Fluor® 488 succinimidyl ester (SE) could be well below 50% by the time of application (according to the manufacturer’s product datasheet). In a number of Alexa Fluor® SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while in all of Biotium’s CF™ dyes the SE group is prepared from an aliphatic carboxylic acid. This structural difference reduces the susceptibility of CF™ dye SE reactive groups to hydrolysis, resulting in relatively stable reactive dyes with consistently higher labeling efficiency compared to other SE derivatives of other fluorescent dyes.

Maleimides, MTS and thiosulfate dyes
Like the succinimidyl ester dyes, these dyes are also susceptible to hydrolysis, although generally to a much lower degree. Thus, for long term storage, anhydrous DMSO is recommended for making stock solutions.

Other reactive dyes
Amines, aminooxy (also known as oxylamine), hydrazide, azide, alkyne, BCN, and tyramide reactive dyes, as well as dye free acids, are generally stable in aqueous solution when stored at -20°C for 6-12 months or longer, as long as no compounds are present that may react with the dye’s functional group. See the product information sheets for specific reactive dyes more information.

Coelenterazines and D-luciferin

Coelenterazines are stable in solid form when stored as recommended; they are not stable in aqueous solution. Concentrated coelenterazine stock solutions (typically 1-100 mg/mL) should be prepared in ethanol or methanol; do not use DMSO or DMF to dissolve coelenterazines, because these solvents will oxidize the compounds. Ethanol or methanol stocks of coelenterazine can be stored at -20°C or below for six months or longer; alcohol stocks may evaporate during storage, so use tightly sealing screw cap vials and wrap the vials with Parafilm for long term storage. Propylene glycol also can be used as a solvent to minimize evaporation. If the solvent evaporates, the coelenterazine will still be present in the vial, so note the volume in the vial prior to storage so that you can adjust the solvent volume to correct for evaporation if needed. Prepare working solutions in aqueous buffers immediately before use. Coelenterazines are stable for up to five hours in aqueous solution.

Aquaphile™ coelenterazines are water soluble formulations of coelenterazines. They are stable in solid form when stored as recommended. Aquaphile™ coelenterazines should be dissolved in aqueous solution immediately before use. They are stable for up to five hours in aqueous solution.

Note that coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

D-luciferin is stable in solid form and as a concentrated stock solution when stored as recommended; it is not stable at dilute working concentrations in aqueous solution. Prepare concentrated D-luciferin stock solutions (typically 1-100 mg/mL) in water, and store in aliquots at -20°C or below for six months or longer. Prepare working solutions immediately before use.

Most of our products are stable at room temperature for many days, but we recommend storage at 4°C or -20°C to prolong shelf life. In the case of many of our aqueous dye solutions, the compounds are very stable at room temperature, but we recommend cold storage to prevent the growth of mold or other microbes over time. Therefore, to save on shipping costs, products with recommended storage at 4°C or -20°C may ship at ambient temperature without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Most of our products are stable at room temperature for many days, so in all likelihood the product will still work just fine. To be on the safe side, we recommend performing a small scale positive control experiment to confirm that the product still works for your application before processing a large number of samples or precious samples.

One exception that we are aware of is GelGreen™, which is more sensitive to light exposure than most of our other fluorescent dyes. If GelGreen™ is exposed to ambient light for a prolonged period of time (days to weeks), its color will change from dark orange to brick red. If this occurs, the GelGreen will no longer work for gel staining.

 

Compound solubility is listed on product web pages, product information sheets or product safety and data sheets. Check the product information sheet for specific instructions for preparing stock solutions. Stock solutions are generally prepared at 10X-1000X the final required working concentration, typically in the range of 1-100 mg/mL. For dyes and indicators, we generally prepare a concentrated stock solution at 5-10 mM in the recommended solvent. The dye can then be diluted to the final desired concentration in the buffer or medium used for the application.

Solubility definitions

DescriptionSolubility
Very Soluble> 1000 mg/mL
Freely Soluble100-1000 mg/mL
Soluble33-100 mg/mL
Sparingly Soluble10-33 mg/mL
Slightly Soluble1-10 mg/mL
Very Slightly Soluble0.1-1 mg/mL
Practically Insoluble< 0.1 mg/mL

To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Difficult to dissolve compounds can be heated to 50°C or higher (note: we do not recommend heating coelenterazine solutions), and vortexed or sonicated until completely dissolved. Some chemical compounds are just kinetically slow to dissolve. This can be especially true when the compounds are highly pure and in crystalline form. An alternative and gentler way to dissolve these compounds can be to simply rock the compound/solvent mixture in the dark overnight.

When preparing stock solutions in organic solvents such as DMF, DMSO, or alcohol for use in living cells or organisms, make a concentrated stock so that the final concentration of solvent in the working solution will not be toxic. A general guideline for immortalized cell lines is to keep the final solvent concentration below 1%; certain cell lines, primary cells or experimental organisms may be more sensitive to solvent toxicity. Similarly, for enzymatic reactions, the final concentration of solvent should be kept below 1% to avoid inhibiting enzyme activity.

Compounds dissolved in aqueous solution can be sterilized by filtration if necessary. Generally, concentrated stock solutions of compounds in organic solvents do not require sterilization, but culture medium can be sterilized by filtration after addition of the compound.

Coelenterazines are predominantly yellow solids, but may contain dark red or brown flecks. This does not affect product stability or performance. If your coelenterazine is uniformly brown, then it is oxidized and needs to be replaced.

Some of our products are packaged from a solution followed by solvent evaporation or lyophilization. If the chemical compound is very lightly colored or colorless and in small quantity, it may become thinly coated on the wall of the vial, making the vial appear empty. So, before you ask for a replacement, please inspect the vial carefully.

To dissolve lyophilized compounds, simply add the appropriate volume of the recommended solvent to the vial to make the desired concentration stock solution, and swirl or gently vortex to mix. Make sure the solvent comes in contact with the inside walls of the vial to fully recover the product.

Note that blue fluorescent dyes such as CF™350, CF™405M, and CF™405S are colorless or very pale yellow, and may be difficult to see.