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FAQs

Mix-n-Stain™ Antibody Labeling Kits

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 (SE) are generally susceptible to hydrolysis, which can result in lower labeling efficiency. Many commercially available fluorescent dyes used for life science research are heavily sulfonated dyes which makes them particularly hygroscopic, worsening the hydrolysis problem. In addition, for several commercially available SE reactive dyes, the SE group is derived from an aromatic carboxylic acid, while the SE group in all of Biotium’s CF® Dyes 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.

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. You can download CF® dye normalized absorption and emission data in Excel format.

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 or with an ice pack. These products may thaw without affecting product performance. When you receive the product, place it under the recommended storage conditions.

Some products are shipped with blue ice packs as an extra precaution against high temperatures. The blue ice packs may be thawed upon arrival without affecting product performance.

Products with recommended storage at ultra low temperature (-70°C) that also ship on dry ice should arrive frozen. If a product you received was shipped on dry ice and thawed during transit, please contact customer service at order@biotium.com.

You can also download our Product Storage Statement here.

For CF® dye, cyanine dye, FITC, biotin, DNP, and dig kits, see the Mix-n-Stain™ Kit Compatibility and Protocol Selection Flowchart, below. We also recommend downloading the updated Product Protocol and completing the pre-labeling checklist to find the right protocol and kit size to use for each antibody you wish to label. Learn more about CF® dyes on our CF® Dye Technology Page, or download the CF® Dye Brochure.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

 

Mix-n-Stain™ CF® Dye Antibody Labeling Kit Compatibility and Protocol Selection Flowchart. Click to enlarge.

 

 

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 35 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.

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 the Product Protocol for more information.

While labeling antibody without stabilizer proteins generally gives the best results, but antibodies with BSA or gelatin can be labeled with Mix-n-Stain™ CF® Dye Antibody Labeling Kits. The BSA or gelatin will also be labeled, but due to the high solubility of CF® dyes, the labeled BSA or gelatin is washed away during standard immunofluorescence staining. We recommend downloading the updated Product Protocol and completing the pre-labeling checklist to find the right protocol and kit size to use for each antibody you wish to label. Also see the Mix-n-Stain™ Kit Compatibility and Protocol Selection Flowchart, below.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

IgG in ascites fluid can be labeled with Mix-n-Stain™ CF® Dye Antibody Labeling Kits with good results using a larger sized kit and our modified Mix-n-Stain™ protocol. Download the updated Product Protocol and complete the pre-labeling checklist to select the appropriate kit size and labeling protocol.

Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols, and cannot be used to label antibodies in ascites fluid. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

Mix-n-Stain™ labeling does not work well for antibodies in serum or hybridoma cell culture supernatant. We recommend purifying IgG from serum or supernatant before labeling.

For CF® dye, cyanine dye, FITC, biotin, DNP, and dig Mix-n-Stain™ kits, 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 the Product Protocol for more information.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then choose a kit size that matches your the amount of antibody you wish to label.

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

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.

The main advantage of Mix-n-Stain™ antibody labeling kits is that they include our 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.

Lightning Link is a registered trademark of Expedeon.

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.

The Mix-n-Stain™ kits do not specifically label the Fc region of the antibody, they will label accessible reactive residues irrespective of the location on the antibody. The image of the labeled dye on Fc region of antibody is merely representative. We design our Mix-n-Stain™ labeling kits to give the optimal DOL for each dye to minimize any effect on the binding affinity of the antibody. Based on this, it is likely that the majority of the labeling is on the Fc region of the antibody, which does not directly bind antigen. There are some cases where antibody labeling can disrupt antibody-antigen binding, usually for monoclonal antibodies, but this is rare based on our experience.

The degree of labeling with Mix-n-Stain™ kits is estimated to be in the range of 4-6 dye molecules per antibody.

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.

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.

Approximately 100%.

For the Mix-n-Stain™ CF® Dye, biotin, Cyanine Dye, FITC, and hapten antibody labeling kits it takes 30 seconds or less to mix the components (antibody, dye and reaction buffer). After another 15 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 in the Mix-n-Stain™ CF® Dye, biotin, Cyanine Dye, FITC, and hapten antibody labeling kits will be complete after 15 minutes. However, a longer reaction time will not adversely affect the labeling.

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.

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.

We now offer Mix-n-Stain™ Nanobody Labeling Kits, specifically designed for optimized labeling of single-domain nanobodies with CF® dyes.

We also have had customers report successful labeling of nanobodies with our original CF® Dye 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. We now offer Mix-n-Stain™ Nanobody Labeling Kits, specifically designed for optimal labeling of nanobodies with CF® dyes.

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.

Our Mix-n-Stain™ kits are designed for labeling antibodies and would not be suitable for labeling oligos. To label oligos with CF® Dyes, please see FAQ Can CF® Dyes be used to label oligos?

Currently, we do not have any products for conjugating DNA to HRP or other enzymes. However, you can order oligos pre-conjugated to HRP from oligo suppliers.

Yes, Mix-n-Stain™ kits can be used to label antibodies containing the preservative ProClin™ 300.

ProClin is a trademark of The Dow Chemical Company.

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.

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.

For CF® Dye, biotin, dig, or DNP  kits, please download the updated Product Protocol and follow the Troubleshooting Checklist for step-by-step troubleshooting of Mix-n-Stain™ labeling and subsequent staining.

Note that Mix-n-Stain™ Maxi 1 mg Scale Kits, Mix-n-Stain™ Fluorescent Protein Labeling Kits (R-PE, APC, PerCP, or tandem dye), Mix-n-Stain™ Enzyme Labeling Kits (AP, HRP, GOx) have different compatibility requirements and labeling protocols. Check the protocol for your kit to verify that your antibody is compatible with labeling, then contact technical support for assistance.

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