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Mix-n-Stain™ Antibody Labeling Kits

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.

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.

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.

 

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.

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