BioLegend FAQs

  • What is the acceptable tolerance range for storage of antibodies and other reagents recommended at 4°C?

    There is an acceptable tolerance range between 2°C and 8°C, for all products recommended to be stored at 4°C.  If your refrigerator at any time has temperatures outside of this range, we recommend you adjust its settings or use a different refrigerator.  

    What is the shelf life of BioLegend products?

    Our products are guaranteed until the expiration date, under proper storage and handling conditions as instructed on our Product Data Sheets.

    What is the epitope of the antibody?

    BioLegend does not epitope map antibodies. Sometimes this information is indicated on the datasheets, if published, but generally, we will not know the exact epitope of the antibody.

    How do I look for reagents that are cross-reactive with a different species (e.g. Rhesus or Cynomolgus)?

    Perform a search for your species using our search box at the top right of the web page. Alternatively, use our advanced search drop down menu for Species Reactivity and select your choice of species.  For a broad overview of all our cross-reactive antibodies, use our Antibody Cross-reactivity Chart.

    What are the concentrations of your antibodies?

    For the antibodies offered by mg, concentrations are typically (exceptions for some products) as follows:

    APC, APC/Cy5.5, APC/Cy7, PE, PE/Cy5, PE/Cy5.5, PE/Cy7, PerCP, PerCP/Cy5.5 = 0.2 mg/ml
    Purified, Biotin, FITC, Alexa Fluor® 488, Alexa Fluor® 647, Alexa Fluor® 700, Pacific Blue™ = 0.5 mg/ml
    LEAF™ = 1.0 mg/ml. For test size products, the concentration can vary by lot, so you may wish to contact tech support, or if you have your lot number, use our look-up tool:

    How do I obtain a Material Safety Data Sheet (MSDS) for your products?

    On each product data sheet there is a link to all of the available MSDS.  Or check our website under "Support". If your product isn't covered in this section, please contact BioLegend Technical Services.

    What is BioLegend's quality policy?

    100% Satisfaction Guarantee. If BioLegend's product does not perform as described on its product data sheet, we'll replace it or refund 100% of the original purchase price, until the expiration date.

    How do I know if an antibody is monoclonal or polyclonal?

    By definition a monoclonal antibody is of one Ig subtype, while a polyclonal antibody contains multiple Ig subtypes.  So look at our product isotype description and if it says: IgG1, IgG2a, IgG2b, IgM, etc, these will be monoclonal, whereas: Goat IgG, Rabbit Ig, etc. will be polyclonal. Most of our polyclonal antibodies also have the term poly in the clone name.

    Can you provide PDF files for the product related journal articles in general or for the ones you cite on the product TDS?

    We have restricted full journal access. Moreover due to copyright issues we will not send full pdf files for the articles we may have. The end user should do their own independent search to see if the article is provided for free elsewhere.

    What is the concentration and expiration date of my antibody lot? Also how can I obtain CoA of the reagent that I received?

    If you already have purchased the product just simply use the concentration lookup, expiration and CoA related web tools on our website (links below) by typing in the lot# (starts with a letter B) associated with the product. If you have not yet purchased the product then please contact Technical Service for details.

    Why are some products discontinued?

    Typically the products are discontinued due to low customer demand or their replacement with in-house produced alternatives. In addition, we do side by side comparison of the the new replacement product with the product that is discontinued to make sure it meets our stringent quality control criteria. We don't guarantee discontinued products.

    How long can you guarantee the stability of antibodies when left in ambient temperature?

    The antibodies are fine (unopened in the original box) for up to two weeks at ambient temperature. That being said, we would recommend protecting dyes from extreme temperature and light exposure. If you have doubts about the antibody, it may be best to test it.

  • Why should I order from a distributor?

    For direct orders shipped outside the U.S., shipping costs typically start at USD $50 for antibody orders and $100 for dry ice shipment. This could vary country to country. Additionally, prepayment is required for direct international shipments. Also, taxes and duties will be paid by the recipient upon shipment delivery. In some countries, the customer needs to apply for an import permit. When ordering from a distributor, the payment terms are more flexible, the customer and technical support is more immediate, and the customer has greater convenience in not having to deal with customs agencies, duties/taxes, and currency exchange.

    Multiple clones available for a particular specificity, how do I choose the right one?

    Check the Product Data Sheets on-line to review applications/characteristics for each clone. If you still have questions, please contact BioLegend Technical Services at:
    * Toll Free Phone (U.S., Canada): 1-877-BIOLEGEND (246-5343) Phone: 858-455-9588 Fax: 858-455-9587
    * E-mail: Click Here

    What is the advantage of ordering per test instead of per mg?

    Antibodies offered per test have been pre-titrated for optimal performance in flow cytometry assays. Antibodies offered per plate have been pre-titrated in ELISA. Cell Biology antibodies offered per µl have been pre-titrated for Western Blot. The antibody packed in an μg format (untitrated) is not necessarily always from the same batch number as a test format (pre-titrated) though the same procedures are performed in the quality control testing. Please also note that not all test formats of the antibodies have lower concentrations than the μg format. It depends on a particular batch tested. If the optimal concentration were determined at 1 μg/test for a particular batch, then it would be 100 μg in the 100 tests. The advantage to using a pre-titrated format is that it provides more convenience for the optimal performance in areas such as flow cytometry assays, especially for beginners who may save time and reagents on titration of the antibody.

    When can I expect to receive my order if I order directly from BioLegend?

    For U.S. orders, if products are in-stock and your order was made before the shipping cutoff time (contact for more info), they are shipped out that day for you to receive the next day. We typically do not ship on Fridays as to avoid packages sitting unattended over the weekend. International direct orders are shipped on Mondays and Fridays, and typically arrive within 2 - 10 days, depending on how long they are in customs.

    How are BioLegend reagents shipped?

    Most products are shipped at ambient temperature.  Our products are sufficiently stable to maintain optimal performance after overnight shipping.

  • What is fluorescence?

    Fluorescence is the emission of light by a substance that has absorbed light. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed wavelength. In flow cytometry, fluorophores are used to tag antibodies in order to provide a signal upon detection of an antigen on the sample when exposed to a single wavelength laser.

    How are Fluorophores conjugated to antibodies?

    There are several different methods that can be used to conjugate fluorophores to antibodies. This can depend on the antibody and type of fluorophore being used. One common method is to conjugate the organic fluorophore via primary amines (lysines) on the antibody. Another method conjugates maleimide-labeled fluorophore with antibody via sulfhydryl groups in the hinge region.

    What is an f:p ratio?

    Fluorescence to Protein (f:p) ratio tells you the average number of fluorophore molecules that are conjugated to each antibody for any given lot of product. These values can vary widely between manufacturers and even among different lots from the same manufacturer. When using isotype controls, it is important to verify that your control has a similar f:p as that of the primary antibody.

    How do I know what buffers I need to use for my flow experiment?

    Be sure to use the buffers recommended in the protocol that you are using.  BioLegend provides a number of flow cytometry protocols, as well as a page to guide you in selecting the appropriate buffers for your flow cytometry experiments.

    What controls should I use for a flow cytometry experiment?

    Isotype controls are recommended for every flow experiment. Isotype controls that do not recognize any known proteins will provide you with information on the background staining of an antibody due to its isotype. For multicolor experiments, fluorescence-minus-one (FMO) controls can help you determine the fluorescence spillover from all your other antibodies and can be used to help in gating positive and negative boundaries. Unstained cells can also provide you with a relative measure of the autofluorescence associated with any particular cell type.

    What is a tandem dye and why do we use them?

    Tandem dyes are fluorophores composed of two distinct fluorophores conjugated together. The resulting tandem uses the excitation property of the donor fluorophore and emission property of the acceptor fluorophore, based on the principles of fluorescence resonance energy transfer (FRET). This allows for novel fluorophores with high stokes shifts (separation between excitation and emission range).

    How does fixation affect fluorophores?

    Fixation with paraformaldyde generally tends to decrease the fluorescence intensity of bound antibodies, particularly with prolonged exposure. This is especially true for nanocrystals and tandem dyes such as those derived from PE and APC. Excessive fixation can also alter the conformation of proteins, causing loss of antibody reactivity to proteins as well.

    How do I know what fluorophores I can use on my instrument?

    Refer to your instrument manual to determine the specifications of your instrument. You may also be able to find specifications in the software provided with your instrument. Note that many instruments are custom built and may not match the standard manual. Also, many instruments have adjustable filter sets, allowing you to configure your instrument to suitably run many different combinations of fluorophores. Always verify that the instrument you plan to use is capable of detecting the fluorophores in your experimental panel.

    What is compensation?

    Compensation is the process of removing spillover (spectral overlap) from other fluorophores into the detector for your fluorophore of choice. For example, due to the wide emission range of FITC, some of the FITC signal “bleeds” into PE giving you false signal in the PE channel. On newer digital instruments, compensation can be applied automatically using single stain controls.

    Does this antibody react with the extracellular domain or intracellular region of the target?

    We don't perform epitope mapping in house. We perform our quality control (QC) test with either surface staining or intracellular staining, as indicated in each product datasheet. When surface staining is performed, it means the antibody recognizes an extracellular epitope. In contrast, if the QC is done with intracellular staining, it means that the protein is expressed in this cellular compartment, but it does not necessarily mean that the epitope recognized is exclusively intracellular. The antibody may still be able to detect the protein if it is expressed or exported to the surface of the cell. It is best to further consult the literature related to the particular clone in this matter.

    Can cells play a part in specific degradation of tandems?

    According to a study (Le Roy C, et al. 2009. Cytometry A. 75:882), APC tandems are degraded through a cell-dependent mechanism.

    Can I swap filters from one laser line to another in a FACS machine?

    If you swap the filter then it may be best that you test the settings with multipeak (6-8) beads such as our Rainbow Calibration Particles and look at their spread with the original filter and recheck the bead spread with the changed filter.

    Do you guarantee that your antibodies are totally pathogen free?

    BioLegend does not test for pathogens in-house aside from the GoInVivo™ product line. However, upon request, this can be tested on a custom basis with an outside, independent laboratory.

    Can I use Maxpar® Ready format clones for flow cytometry staining?

    We have not tested the Maxpar® Ready antibodies formulated in solution containing EDTA for flow cytometry staining. While it is likely that this might work in majority of the situations however it is best, however, to use the non EDTA formulated version of the same clone for flow cytometry testing. Presence of EDTA in some situations might negatively affect staining.

    Is there a difference between buffer formulations related to Maxpar® Ready and purified format antibodies?

    The Maxpar® Ready format antibody clones are formulated in Phosphate-buffered solution, pH 7.2, containing 0.09% sodium azide and EDTA. The regular purified format clones are formulated in solution that does not contain any EDTA. Both formulations are however without any extra carrier proteins.

  • What is the difference between test and µg size?

    The test size products are pre-titrated for optimal staining of 1 million cells in 100 µl volume.  On the other hand, µg size products are at a defined concentration regardless of the optimal usage.  It is still really important, regardless of format, to use the correct isotype control at the same amount (µg) as their antibody of interest.  If the concentration of the antibody is not on the vial, then call us or email (by clicking on “More Info” below) to obtain the concentration for your lot of product.

    What is the difference between the FC and ICFC formulation? Are there different optimal concentrations for each?

    FC is for cell surface staining and ICFC is for intracellular staining.  Yes, the concentrations can be different. The IC antibodies and isotypes have optimized fluorochrome conjugation properties to give optimal performance for IC staining.

    Can I use less volume or less cells than what is recommended per test?

    Yes, you can use less cells or less volume per test, as long as the antibody concentration does not change.  For example, if you decrease the staining volume from 100 to 50 µl, you could use half the amount of antibody.  But, if you decrease your cell numbers from 1 million to 100,000 cells, and your staining volume is still 100 µl, then you should still use the data sheet recommended amount of antibody.

    I see there are multiple clones for the product I want, which one do I choose?

    If you are not looking for a specific clone check we recommend to use the most popular clone based on Pubmed or Google Scholar.  This will give you an idea on how often a clone is used compared to another.

    Can I use BioLegend antibodies for cell sorting?

    In general our antibodies can be used for cell sorting, but we do not routinely test this in house.  Please note the fluorochrome conjugates are not endotoxin tested and contain 0.09% azide. Typically this concentration is low, so the azide amount will not affect viability of the cells.  As for activation of cells, typically the incubation time with the antibodies for staining is short (~20minutes) and this should not cause activation.  Incubation of antibodies with cells at 4°C or on ice will help prevent activation.

    What is the F/P ratio for my fluorochrome conjugated antibody?

    All PE, APC and their tandem dyes labeled antibodies have a F/P of 1:1.  Other formats such as FITC or Alexa dyes have various F/P ratios, typically within range of 3-7

    Can I use the antibody at a lower concentrate/volume than what is recommended?

    It is recommend to perform a titration because sometimes you find you can use less antibody for your own experimental needs, and also to have a feel for how the antibody behaves in your own hands in your particular experiment.

    How does BioLegend choose the optimal concentration of use of an antibody in flow cytometry?

    Generally, we test 4 to 6 dilutions with the most commonly used target cells (if they are available) for the titration curve, then determine the optimal concentration based on the S/N (Signal/Noise) ratio. 

    Can I freeze your flow antibodies?

    We do not recommend freezing our antibodies as this can denature the antibody or cause fluorochromes to uncouple from the antibody during freeze/thaw. In addition, the antibodies can clump and form aggregates, allowing it to bind nonspecifically to cells. We recommend keeping our flow antibodies at 4°C, protected from light.

    What concentration/amount of the isotype control should I use?

    Be sure that the isotype control matches the same concentration/amount being used for the primary antibody. Concentrations between isotype and primary antibodies are not always identical. Therefore, using the same volume may not produce equal amounts of antibody.

    I want to stimulate my cells for intracellular cytokine staining. What do you recommend?

    Can I use PE labeled antibodies for studying murine plasma cells?

    According to a study, PE conjugated antibodies, but not FITC or APC-antibodies, selectively stain lamina propria plasma cells (LP-PCs) from the murine small intestine after membrane permeabilization. This phenomenon was also observed in plasma cells isolated from bone marrow, spleen, and mesenteric lymph nodes. Therefore, beware of this potential effect before using PE labeled antibodies for intracellular staining of murine plasma cells.

    Can I preincubate my primary and secondary antibodies?

    Theoretically you can if the secondary antibody binds to the Fc portion of the primary. However if the secondary also binds to the light chains then it is possible that it may interfere with the ability of the primary antibody to bind to its target. Therefore it needs to be tested.

    Since cyanine-based dyes have propensity to non specifically bind to monocytic population, should I use cynanine dye based fluors for my monocytic population phenotyping?

    This non specific binding should be kept in mind. You can try testing the antibody first to see how much non specific binding you see in your sample. It is also best to follow routine steps such as Fc blocking and titration of the antibody in order to reduce background while doing flow cytometric staining. You can also consider our True-Stain Monocyte Blocker™ to see if it improves staining (Cat. No. 426101).

    Do tandem dyes vary between sources and lots?

    No two tandem dyes are created equal. For the same tandem there may be differences between vendors and even between lots. For consistency it may be best to stick with the same source and lot if possible throughout a single study.

    As a hypothetical example, can I use the same tandem dye conjugate but different antibody specificity for setting up compensation controls?

    Since tandems can exhibit variation between sources and lots, it is best to use the exact same antibody as a compensation control. It may be best to use compensation beads for setting up the compensation.

    Can a particular clone work on fixed cells?

    If the product web sheet for that clone does not reveal this information then it is possible that we have not tested the fixation compatibility of that particular clone. Clone specific literature search is advisable in such situations. You can go to to learn more.

    Do you sell just the fluorescent dyes separately?

    We don't offer fluorescent dyes separately. However we do offer custom conjugation services. Please contact oursales department for quotes.

    Do you offer conjugation kits?

    We don't offer dye conjugation kits.

    I am not getting consistent staining while comparing two different antibody clones from different sources for the same target. What may be the issue?

    Differences in characteristics of antibody clones such as whether they are polyclonal or monoclonal, as well as the source are variables that may lead to inconsistent staining. The difference may be particularly pronounced if a monoclonal antibody is compared with a polyclonal antibody for the same target. Even if the two antibodies are monoclonals, it is possible that their staining pattern may look different. It may be best to search relevant literature.

    Where can I make a custom request for bulk orders, custom conjugation, or custom antibody production?

    Do you sell cell lines you use to QC your flow cytometry products?

    We don't offer cell lines. American Type Culture Collection may offer further guidance on this issue.

    What markers would you recommend for my population of interest?

    Please refer to our Cell Marker webpage. For a more streamlined approach, check out the Essential Markers webpage. It is also advisable to consult the literature to find out further information for a particular cell type.

    Can I keep my blood sample (drawn in the presence of EDTA) for later analysis by flow cytometry?

    It is advisable to use EDTA blood within 24 hours.

    I didn't obtain any signal with your fluorescent antibody using compensation beads?

    When using compensation beads please double-check the beads specific details such as exceptions related to certain antibody light chains or host species that may not be compatible with a particular set of commercial beads.

    How do you determine the antibody concentration?

    We use absorbance (OD method). Concentration = OD280*DilutionFactor/1.4 (extinction coefficient).

    Is the competitor's fix/perm buffer systems compatible with your antibodies for applications such as flow cytometry?

    We have not validated various competitors' fix/perm buffer systems in house so it is empirical to find out if the combination works. It may be best to stick with the buffer system that is validated for a particular clone.

    Is the Fc blocking step required for intracellular staining?

    Typically it is not required, but if surface staining is involved as part of the staining then do add an Fc blocking step. 

    What type of PE do you use in your conjugates?

    We use R-PE in our conjugates.

    Does sodium azide and carrier protein such as serum or BSA interfere with conjugation?

    Sodium azide up to 0.09% w/v does not hinder Amine-Reactive or Sulfhydryl-Reactive conjugations. On the other hand, carrier proteins such as serum or BSA interfere with such conjugations.

    How does pH and staining temperature affect Annexin V-Phosphatidylserine binding?

    Annexin-Phosphatidylserine binding is lost below pH 5.2 and with prolonged incubation over a temperature of 42°C.

    What is the concentration of Human TruStain FcX™ (Fc receptor blocking solution, Cat#422301)?

    This information is proprietary.

    Is our Human Trustain FcX™ (cat# 422302) compatible with anti human CD16, CD32 and CD64 clones 3G8, FUN-2 and 10.1 respectively?


    Can I use common compensation control for GFP, CFSE and FITC because they emit in the same channel?

    It is not recommended even if they emit in the same channel because these are still different fluors with different brightness intensities. Individual compensation controls should be employed.

    I am unable to see expression of T cell markers such as CD3 and CD4 post activation.

    TCR-CD3 complexes on the T-lymphocyte surface are rapidly downregulated upon activation with peptide-MHC complex, superantigen or cross-linking with anti-TCR or anti-CD3 antibodies. PMA/Ionomycin treatment has been shown to downregulate surface CD4 expression. Receptor downregulation is a common biological phenomenon and so make sure that your stimulation treatment is not causing it in your sample type.

    How many biotin molecules are per antibody structure?

    We don't routinely measure the number of biotins with our antibody products but the number of biotin molecules range from 3-6 molecules per antibody.

    What is the F/P ratio range of our BV421™ format antibody reagents?

    It is lot-specific. On average it ranges between 2-4.

    Does staining at RT or even at 37°C help for checking chemokine receptors expression?

    Due to continuous recycling of many chemokine receptors, it may be worthwhile to consider staining at RT or at 37°C if the staining at lower temperature (which can potentially reduce receptor turnover) is not optimal.

    How stable is PerCP/Cy5.5 tandem as compared to PerCP alone?

    PerCP/Cy5.5 is quite photostable and also better than PerCP alone in withstanding fixation.

    Why does your Anti-Neu5Gc kit (cat# 146901) come with its own blocking solution? Can I use my own?

    Many common blocking solutions contain reagents such as FBS that would contaminate your samples with Neu5Gc, thus leading to generation of background. The blocking solution in our kit is guaranteed to be free of any of these contaminants.

    Have you tested this LEAF™/Ultra-LEAF™ antibody for in vivo or in vitro applications?

    We don't test our antibodies for in vivo or in vitro bioassay type testing unless otherwise indicated on the technical data sheet. It may be best to further consult the literature to find clone specific information.

    Is your Foxp3 buffer set compatible with anti mouse and/or human cytokine antibodies?

    Nuclear permeabilization generally creates larger pores in a cell that can allow cytokines to leak out. In order to find out whether this occurs, you would have to run a sample fixed and stained with a non-nuclear permeabilizing buffer (such as Cat. No. 421002).

    Can I stain whole blood with anti-FOXP3 using your Foxp3 staining kit?

    It is not recommended. It is best to use PBMCs for this testing.

    Can your Nuclear Factor Fixation and Permeabilization Buffer Set (cat# 422601) and FOXP3 Fix/Perm Buffer Set (cat# 421403) be used in place of each other?

    You have to try it in your system but it should work.

    Why there is no good correlation between intracellular cytokine staining and ELISA?

    Intracellular cytokine staining for flow cytometry represents a snapshot in time and ELISA represents cytokine accumulation overtime.

    Why it is hard for flow cytometry or ELISA compatible antibodies to work for western blot?

    The antibodies that are tested via flow cytometry or ELISA work by recognizing 3D target conformation and epitopes. Harsh treatment associated with WB such as boiling leads to linearization of the target epitopes, which may then lead to non recognition of the target.
  • What is Brilliant Violet™?

    Brilliant Violet™ is a family of highly fluorescent polymers, excitable by the 405 nm violet laser, created by Sirigen based on Nobel Prize winning chemistry. There are seven fluorophores available: BV421™, BV510™, BV570™, BV605™, BV650™, BV711™, BV750™, and BV785™. Each has a unique emission spectra as well as a unique set of advantages over other 405 nm excitable fluorophores on the market. Brilliant Violet™ fluorophores are suitable for surface or intracellular flow cytometry, providing excellent signal with very little background. For excitation/emission and beta testing data, recommended filters, and comparable flourophores, as well as guidance on the strengths of each member in the family and incorporating them into multicolor panels, see our dedicated Brilliant Violet™ page.

    How does Brilliant Violet™ perform compared to other fluorochromes?

    Brilliant Violet 421™ antibody conjugates give an exceptional signal-to-noise ratio, in some cases greater than 10-fold better than Pacific Blue™.  It gives PE-equivalent brightness or better on the violet laser.  Brilliant Violet 510™ is a non-tandem polymer, and provides improvements over Pacific Orange™, AmCyan, and Horizon™ V500. Brilliant Violet 570™ antibody conjugates also provide much improvement over Pacific Orange™, by as much as 6-fold.  Brilliant Violet 605™ and Brilliant Violet 650™ are significantly brighter than eFluor® 605 and eFluor® 650, respectively, by as much as ten-fold, but are not much brighter than Qdot® 605. Brilliant Violet 711™ is significantly brighter than eFluor® 700 and eFluor® 650, by as much as ten-fold, but is not much brighter than Qdot® 705. Brilliant Violet 750™ does not currently have a comparable equivalent, but rates a 3 out of 5 based on our brightness staining index. Brilliant Violet 785™ is similar in brightness to Qdot® 800. Since BV605™, BV650™, BV711™, BV750™, and BV785™ are non-nanocrystals, they are likely to perform better for intracellular staining and are not affected by fixation.  Learn more

    Can Brilliant Violet™ be used for microscopy?

    Yes, Brilliant Violet 421™ is exceptionally bright and photostable, making it an excellent choice for microscopy. View the beta-testing data

    What bandpass filter should I use to detect each Brilliant Violet™ fluorophore?

    We recommend using the standard 450/50 filter to detect Brilliant Violet 421™, which is the same filter used to detect Pacific Blue™.  We recommend using the 510/50 filter to detect Brilliant Violet 510™. For Brilliant Violet 570™ we recommend the 585/42 bandpass filter, commonly used for Pacific Orange™. For BV605™ detection, we recommend the standard Qdot® 605 filter, 610/20 with a 595LP dichroic. For BV650™ detection, the standard Qdot® 650 filter, 660/20 with a 630LP dichroic, is recommended. For BV711™ detection, we suggest the standard Qdot® 700 filter, 710/50 with a 685LP dichroic. For BV750™ detection, we suggest using a 740LP dichroic with a 780/60 bandpass filter. For BV785™ detection, we suggest the standard Qdot® 800 filter, 780/60 with a 750LP dichroic. See the Fluorescence Spectra Viewer for complete excitation and emission data.

    Can I use all Brilliant Violet™ dyes together?

    If you plan to use all Brilliant Violet™ dyes, including BV750™, you will require a flow cytometer capable of spectral unmixing or a decagon configuration off the violet laser to accommodate all the fluorophores. If you’re not using BV750™, then, yes, the rests of the Brilliant Violet™ fluorophores can be detected by an appropriate configuration off an octagon PMT set-up off the violet laser. For the best result, a balance of PMT voltages is beneficial whereby one PMT should not be significantly different from others. Imbalanced PMT voltage situation can potentially amplify the spillover from one fluor to the another channel. Also note that pre-mixing of the Brilliant Violet™ dyes before staining for an extended time can lead to dye-dye interactions.

  • Why is BioLegend entering this agreement with Covance?

    BioLegend is seeking to expand our product portfolio into new areas including Neuroscience, Immunopathology, and IHC reagents. Covance Antibodies not only expand BioLegend's catalog, but also complement current BioLegend offerings.

    Why is Covance selling the Covance Antibody group to BioLegend?

    The goal of divesting the group was to place it in an organization that better aligns with the focus of the group, would value and grow the group to increase the business. We believe that BioLegend's commitment to providing researchers with the most comprehensive and cutting-edge high quality reagents for life science research, strong background in research tools, and aggressive product development program positions the Antibodies Products group for increased commercial growth and greater career opportunities for employees.

    Will management of the Antibodies Product group remain the same?

    Yes, the entire staff of the Antibodies Product group remains the same.

    I get a great discount for my BioLegend products, can I apply this to former Covance Antibody products?

    We are always looking to provide our products at an outstanding value. At the moment, we are not extending any current BioLegend discounts to former Covance products. We are working to realize additional process efficiencies through our integration efforts and will, as always, look to share the benefits of such with our customers.

  • I am going to do a functional assay. Which grade of purified antibody should I choose?

    BioLegend's LEAF™ (Low Endotoxin, Azide-Free) purified antibodies are specifically designed for functional analyses, providing the most accurate results with minimal negative effects. LEAF™ purified antibodies are tested for sterility, and endotoxin is <0.01 ng/µg of antibody. For bulk amounts of functional and pathogen-tested antibody, consider our GoInVivo™ products.

    Does BioLegend test each LEAF™ antibody by functional assay?

    No, BioLegend does not test LEAF™ antibodies by functional assays. Due to the possible complexities and variations of uses of biofunctional antibodies in different assays and because of the large product portfolio, BioLegend does not currently perform functional assays as a routine QC for the antibodies unless otherwise indicated. However, we do provide references in which the antibodies were used for functional assays and we do perform QC to verify the specificity and quality of the antibody based on our strict specification criteria.

    Does BioLegend test each LEAF™ antibody for potential pathogens?

    No, BioLegend does not test for pathogens in-house.  However, we can recommend an outside vendor to perform this testing as needed.

    Does BioLegend manufacture antibodies at GMP grade?

    BioLegend antibodies and reagents that carry the IVD or ASR designation are manufactured in a registered GMP facility, while our other products are manufactured under ISO 13485 certification. Both environments provide high quality products. To learn more about our quality standards visit our Quality Control page at:

  • How do I choose the right isotype control for the primary antibody I'm using?

    Refer to the for listed isotype and format of each primary antibody and match the isotype control appropriately.  For example, if you have a PE conjugated primary antibody with Rat IgG1, k isotype, use PE Rat IgG1, k isotype control Catalog # 400407 or 400408, as your isotype control. For your conveniece, most products have their isotype control listed under their "related products" or directly hyperlinked under the "isotype" field.

    How do I know how much isotype control to use for test products?

    Your vial has the antibody concentration on the label, in some cases.  If not, call or email tech support to (click "More Info" below) and ask for the concentration of your lot of antibody.   Match your isotype to the concentration of the primary antibody. Or, use our lookup tool:

    Can rat IgG1, lambda isotype be substituted with rat IgG1, kappa as an isotype for flow cytometry?

    Yes, you can since kappa and lambda represent light chains which don't contribute to the background staining.

    Is it possible to use an isotype control recommended for ICFC for cell surface staining control?

    It is best to use ICFC validated isotype control for ICFC staining and surface stained validated isotype control for surface staining.

    IgG2a gene is deleted in some mouse strains, which ones are they?

    IgG2a gene is deleted in C57Bl/6, C57Bl/ 10, SJL, and NOD mice. It is replaced with IgG2c gene instead.
  • What is the difference between ELISA MAX™ Deluxe, ELISA MAX™ Standard, and LEGEND MAX™?

    LEGEND MAX™ kits with Precoated plates : Fully validated KIT

    • Most convenient, analytically and biologically validation provided, shortest protocol
    • Ready to use reagents, including Pre-coated plates

    ELISA MAX™ Deluxe: 

    • Cost effective, includes some buffers, plates not included
    • Pre-titrated antibodies, AV-HRP, Recombinant Standard, Coating Buffer, Assay Diluent, and TMB Substrate Reagent.

    ELISA MAX™ Standard: Cost effective development SET

    • Most cost effective, plates not included
    • Pre-titrated antibodies, AV-HRP, and Recombinant Standard.
    • Optimization required


    Do the ELISA MAX™ Deluxe Sets come with plates?

    No, the ELISA MAX™ Deluxe sets no longer come with plates, but Coating Buffer and Assay Diluent (for blocking and dilutions) are included in the ELISA MAX™ Deluxe Sets. Plates can be ordered separately (Cat. No. 423501), and instructions on coating the plates are in the Sets' product manuals and website under "Support".

    Can I mix reagents from different ELISA MAX™ Sets, or use them for other applications?

    This is not recommended. The ELISA MAX™ reagents are optimized for a particular set lot, and they are not recommended for applications other than ELISA.

    What is the shelf life of BioLegend ELISA products?

    BioLegend's LEGEND MAX™ Kits are guaranteed for 3 months from the date of receipt. ELISA MAX™ sets are guaranteed for 12 months from the date of receipt. For lot-specific expiration date, refer to the box label on each Kit or Set.

    How should the plates be stored after coating?

    If coated plates cannot be used immediately, they should be sealed and stored overnight at 4°C.

    What kind of coating buffer do I use for my ELISA?

    This may be dependent on the cytokine being detected, but in general scientists use PBS, pH7.4 or carbonate buffer at pH 9.5.

    Why do we recommend not to use azide in ELISA buffers?

    Azide is an irreversible inhibitor of HRP.

    When should I use LEGEND MAX™ ELISA kits with pre-coated plates?

    LEGEND MAX™ Kits are ready-to-go, designed for ease-of-use, minimizing requirements for coating the ELISA plates and preparing buffer dilutions.  We recommend LEGEND MAX™ Kits for users who are new to ELISAs or are short on time.

    Can I use different components from different companies for my ELISA?

    Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected. Recombinant standards used are different. First of all, different kits may use recombinant proteins expressed and purified using different methods. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities. Each BioLegend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols can affect the final assay performance.

    How can I obtain better signal and sensitivity for my ELISA assay?

    • Increase incubation times (1st incubation, detection, avidin-HRP or TMB substrate)
    • Shake plates during incubation steps
    • Make sure that the standard is completely reconstituted before use.
    • Increased washing and soaking in between washings to further decrease background.
    • If possible read the plate at 570-590 nm for background subtraction.
    • Improve duplicate CV% by controlling pipetting error, washing with bigger volume of washing buffer, etc
    • Use a 5-PL or 4-PL curve-fitting method for better calculation at the lower end of the curve. This is usually done with a better curve-fitting software, rather than the linear curve fitting.

    Should I use serum or plasma samples for my ELISA experiment?

    This is dependent on the targets being detected and the biological processes. Customers are advised to study the difference between serum and plasma for the targets of interest and decide on the sample type to be used for quantification. Depending on targets, there may be a difference in concentrations of the targets between serum and plasma. The most important factor in preparing plasma or serum samples is consistency in preparation to ensure precise measurements. In general, plasma/serum samples should be free of particulate matters, contain no excess lipids, and have no hemolysis.
    These types of contaminants will contribute to background, and adversely affect the precision of the assay. The key is to prepare the sample the same way each time. That is, centrifuging samples at the same speed, for the same time, removing the serum or plasma immediately after centrifugation and aliquoting and freezing the samples in the same time. Avoid repeated freezing and thawing cycles.

    Why are my sample cytokine concentrations different on the LEGEND MAX™ kits vs other Elisa kits?

    Currently, it is very difficult to  obtain exactly the same  sample concentrations at pg/ml levels when comparing  different cytokine kits from different vendors, partly because of following reasons:
    • As mentioned in the question above, the immunoreactivity for the reagents vary from different suppliers.
    • In the area of cytokine quantification particularly for those cytokines at pg/mL ranges, the general consensus is that it is more important to have matching biological trend instead of matching absolute concentrations.  This is to say that the same cytokine measured by different kits from different vendors should show the same pattern of biological changes predicted for the relevant biological treatments.
    •  For some cytokines, it is not uncommon  that  the cytokine concentrations vary a few-fold between kits for the reasons mentioned above.
    • It is critical that kits produced by the same vendor  to keep its assay products consistent from lot-to- lot over time.
    We tested  ELISA standards from competitor's kits. Results showed clear discrepancies in the relative potency of the standards in term their immunoreactivities.

    Can I use your ELISA kits for my tissue samples?

    Almost all of BioLegend’s ELISA products can be used for tissue/ cell extract/homogenate samples as long as the samples are prepared in such a way that they are compatible with immune-reactivity:

    • Tissues/cells should be lysed or homogenized in a neutral pH buffer that contains no denaturing chemicals (such as urea, thiourea, SDS).

    • No or minimal levels of detergent (SDS, Triton X-100 etc).

    • No excessive ionic strength (salt concentration greater than physiological ionic strength).

    • The buffers should contain sufficient protease inhibitor cocktails to preserve the target proteins from proteolytic degradation by enzymes released from cells.

    I ran out of some ELISA kit components, can I buy separately?

    Yes. Please contact and provide lot information of the kit and its components.

    What antibody clones are used in your ELISA Kits?

    That information is proprietary. However the clonality (polyclonal or monoclonal) and host species details may be provided upon request.

    For some of your ELISA kits, why do my serum samples require dilution with assay buffer?

    Dilution with assay buffer is required to minimize the matrix difference between the samples and the standards to achieve better accuracy.

    What is the sensitivity of your ELISA kit?

    For ELISA MAX™ Standard format the sensitivity should match the ELISA MAX™ Deluxe version if BioLegend components are used. As for individual LEGEND MAX™ and ELISA MAX™ Deluxe formats the sensitivity values are mentioned in the manual for each kit.

    What is the expected concentration of a particular analyte in biological samples (e.g. in serum samples of naïve animals or normal humans)?

    Since every sample is unique, it is difficult to predict as this may depend on the sample preparation and the nature of the analyte. For LEGEND MAX™ kits refer to the respective ELISA manual for more information.

    How many samples can I run with your kit?

    It depends on how your samples are analyzed whether in duplicate or triplicate. For example you can run 80 samples with no replicates or 40 samples in duplicates and so on.

    Can samples such as serum be reused?

    It is recommended that for accurate results samples be stored aliquoted for one-time use only. However it is empirical to find out if reusing samples work for a particular analyte as it will depend on sample stability.

    Can coating with capture antibody be carried out for longer than overnight?

    It is possible. Generally, coating for 16-20 hours at 4°C is recommended. Longer incubation time may increase the amount of capture antibody bound to the plates and this may also increase the background noise.

    At what step can I delay my ELISA procedure for a few days? Also can I freeze my plates for later use?

    It is best to follow the recommended protocol without unnecessary delays. However if you wish to delay then it may be better to delay the procedure at the blocking step (after coating with the capture antibody). You can add 200 μl of blocking buffer in the wells and either the plates can be kept overnight at 4°C (minimal or no loss of signal) or frozen at -20°C for few days (it may have some impact on the signal). Delaying the procedure at the first step (coating with capture antibody beyond 16-20 hrs at 4°C) is not advisable because it may lead to high background.

    Can I use tissue culture grade sterile plates for ELISA?

    No. Tissue Culture grade plates are designed for cell culture purpose and do not typically have high binding capacity. We recommend using high protein binding plates such as Nunc Maxisorp™ plates (Cat# 423501).

    Can I add an extra standard at the lower end of the standard curve to enhance the sensitivity of my assay?

    We don’t recommend this. Sensitivity of a kit depends on the individual components and their collective validation as a kit and it will not change by adding extra points to the standard curve.

    Can I add an extra standard at the higher end of the standard curve in order to determine higher concentration of the analyte?

    While this may be possible, you may end up with a plateau in signal at higher concentrations of the standard. It is generally recommended to use the concentration range recommended.

    Can I use your matched ELISA pair antibodies with protein standard from another company?

    Theoretically you can but we don't recommend it and we also can't guarantee the performance of the kit as indicated in the product manual. Antibodies may not recognize or show poor binding towards the protein standard if the immunogen protein used to generate these antibodies is different in terms of structure or sequence. It is therefore empirical to find out if it works. It is best to acquire the ELISA kit components from one source.

    Can I use the recombinant standard provided with the kit for bioassay?

    No. It is not recommended because the ELISA protein standards are not sterile, may contain other carrier proteins and sodium azide, and are not tested for bioactivity. Therefore this material is not bioassay grade.

    Can I use the capture and detection antibody provided as part of an ELISA kit for other applications such as flow cytometry staining?

    Since the antibodies are validated in house for ELISA, it is empirical to find out if the antibodies work for applications such as flow cytometry. We recommend you use flow cytometry validated antibodies for this purpose.

    Does phenol red in the medium interfere with an ELISA assay?


    I am using the biotin and purified formats of the same antibody clone to try to set up my own ELISA, but I’m having no success.

    If you are using the same monoclonal clone for both the detection and capture antibodies, the epitope may already be occupied by one of the antibodies and prevent binding of the other. You should always choose different monoclonal antibodies for capture and detection. If a polyclonal is used, it may serve as both the capture and detection antibodies since polyclonals can recognize different epitopes.

    In your LEGEND MAX™ ELISA Kits, there is a step that calls for a washing of the plates before even adding any sample to it. What is the purpose of this step?

    We typically use a stabilizer for pre-coated plates. The washings were designed to remove these components before you start the assay. If you do not do the washings, the effect on assay performance is negligible.

    We ran out of capture/detection antibody in our ELISA kit. Can we use a standalone/single antibody to replace it?

    No, we don’t recommend it.

    I have multiple LEGEND MAX™ ELISA kits that I want to run simultaneously. Can I use the same wash buffer for all the kits?

    The Wash buffer is the same for all the current LEGEND MAX™ kits. All the part numbers on the Wash Buffer bottles in these kits should be the same. For ELISA MAX™ Deluxe and ELISA MAX™ Standard sets, we provide a recipe for the wash buffer on each kit’s technical data sheet. This recipe is the same for all ELISA MAX™ sets.

    I ran out of assay diluent provided with ELISA MAX™ Deluxe kit.

    You can order more assay diluent Cat# 421203.

    What is the difference between your two substrate solutions TMB Substrate Reagent Set (Cat#421101) and TMB High Sensitivity Substrate Solution (Cat# 421501)?

    TMB Substrate Reagent Set (Cat# 421101) contains two components, which should be mixed immediately prior to use. The TMB High Sensitivity Substrate Solution (Cat# 421501) contains everything in one solution with special stabilizers and is a high kinetic substrate solution which generally gives higher signal. The type of substrate used for an assay can affect the optical density (OD) achievable and the time required to achieve the desired OD.

    Why there is no good correlation between intracellular cytokine staining and ELISA?

    Intracellular cytokine staining for flow cytometry represents a snapshot in time and ELISA represents cytokine accumulation overtime.
  • My standard curve is not linear; can I use the non-linear part of the standard curve during analysis?

    Yes. It is possible to use the non-linear part of the standard curve for calculation of results. The LEGENDplex™ data analysis software uses a five-parameter curve-fitting algorithm, which determines the minimum and maximum detection concentrations of each target and reports them. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and reanalyzed.

    What is the shelf life of LEGENDplex™ kits?

    LEGENDplex™ kits are manufactured with a 2 year shelf-life and are guaranteed for a minimum of 6 months after the date of receipt. Specific expiration dates can be found on the product label on each LEGENDplex™ assay box.

    Should I perform the assay with the filter plates or with V-bottom plates?

    Filter plates or V-bottom plates have been included in some kits for your convenience. Performing the assay with filter plates is generally recommended. A vacuum filtration unit is required to work with the filter plates. If however, you don’t have access to a vacuum manifold, then you can use the V-bottom plates or micro-FACS tubes as described in the manual and follow the recommended assay protocols for the type of plates or tubes you choose. All tubes and plates should be made from low binding polypropylene. Polystyrene ELISA or cell culture plates should not be used.

    The kit instruction manual says that the kit is validated for serum, plasma, and culture supernatant sample types. Can I also use tissue homogenates with LEGENDplex™?

    Yes, all the above sample types may be used with LEGENDplex™ kits as long as they are prepared in a manner compatible with immunoassays. Tissue homogenate samples should be prepared in a neutral pH buffer containing no denaturing chemicals (e.g. SDS, urea, thiourea, cholate, etc.), no ionic detergents, and minimal amounts of non-ionic detergents (e.g. NP-40). The homogenization buffer should avoid excessive ionic salt concentrations (i.e. above physiological levels), and should contain protease inhibitors. In addition, the samples should be centrifuged after lysis in order to remove particulates.

    What curve-fitting algorithm can I use for generating my standard curve?

    The LEGENDplex™ data analysis software uses a default 5 parameter logistic curve-fitting algorithm, which determines sample concentrations and calculates minimum and maximum detection concentrations of the standard curve. If the default algorithm does not work for a particular set of data, other curve fitting methods are available in the Options of the software. If sample concentrations fall outside of the maximum detectable range, the sample will have to be diluted and re-analyzed.

    What is the difference between LEGENDplex™ and Luminex® Assays?

    LEGENDplex™ and Luminex® are both bead-based multiplex immunoassays that use the basic principles of sandwich immunoassays. Both systems use fluorescence-coded beads to achieve multiplexing. The major difference is in the data acquisition. LEGENDplex™ uses common lab flow cytometers for data acquisition, whereas Luminex®-based assays have dedicated machines for this purpose. Therefore, one of the advantages is that LEGENDplex™ can be run on widely available flow cytometers without needing specialized machines. As such, LEGENDplex™ cannot be run on Luminex® machines.

    What are the compatible flow cytometers for the LEGENDplex™ assays?

    LEGENDplex™ assays can be used on most commonly used flow cytometers that can read APC and PE, such as BD FACSCalibur™, BD FACSCanto™, BD FACSCanto™ II, BD LSR I™, BD LSR II™, BD LSRFortessa™, BD FACSAria™ I, II, III, BD Accuri C6™, BD FACSVerse™, Gallios™, CytoFLEX™, Moflo XDP™, Attune™NxT, NovoCyte™, MACSQuant®, and Guava® easyCyte. Please refer to the “Materials to be provided by end-user” section of your LEGENDplex™ manual for details on the requirements of the machines in terms of laser and channel configurations. For other brands of flow cytometers, the end-user needs to make sure that the machine is set up properly before use.

    When do I need to do instrument compensation?

    If a single laser instrument is used for both reporter (FL2 or PE) and classification (FL3), then compensation is needed to resolve the signal spillover from FL2 to FL3, and vice-versa. Even on dual laser instruments, some compensation may be required. Please check your machine for the need for compensation and when required use the calibration beads provided and follow the Flow Cytometer Setup instructions in the LEGENDplex™ manual. In addition, adjust the PMT voltages such that there is good bead separation, the bead populations are not out of scale, and the PE signal is appropriately amplified to ensure proper signal sensitivity.

    Is special software required for data analysis?

    Typically, flow cytometers generate output files in FCS format (e.g. FCS 2.0, 3.0, or 3.1), and in some cases, in list mode file format (LMD). FCS files can be analyzed by different software. Data generated from using LEGENDplex™ kits can be analyzed using the LEGENDplex™ data analysis software freely available for downloading at the BioLegend website ( A free-of-charge software license dongle is provided in the kit (required for PC users). The Mac software is available for use with a file license.  Please check our website for most updated versions of the software.

    After I finish the staining process, how long can I wait before reading my LEGENDplex™ samples?

    The plate or tubes containing the bead assays can be re-suspended in Wash Buffer, kept overnight at 4˚C while being protected from light, and be read the next day. There may be a decrease in signal, but overall, the assay results should not be significantly affected. Storing the samples for extended periods of time is not recommended, as it could lead to further reductions in signal.

    If I don't have a vacuum, how do I remove the liquid from my plate?

    Do NOT invert the plate to dump the liquid. The beads will not adhere to the plate. After centrifugation using a swing bucket rotor with a plate adaptor, you can remove the liquid by using a pipette.

    Does LEGENDplex™ software run on Apple Macintosh?

    Yes. We now have software for both Macs and PC. Get more details and download the software at:

    The biological samples I used in my LEGENDplex™ assay are BSL 2+. Can I fix them prior to flow cytometer acquisition in order to sterilize my samples?

    We have found that fixing/sterilizing in samples 1-4% paraformaldehyde in 1X PBS prior to reading the samples out on a flow cytometer is compatible with our LEGENDplex™ reagents. However, incubation time with PFA should not exceed 10-15 minutes. Immediately following the fixation, the PFA should be washed out using the 1X Wash Buffer supplied with the kit. Beads should be resuspended in the 1X Wash Buffer supplied with the kit. Prolonged incubation of beads with 1-4% paraformaldehyde will decrease the signal dramatically.

  • What is the level of variability from one experiment to the other?

    If the protocol is followed closely, the variability should be minimal. The variability should be similar to single vial antibody staining.

    How should the kit be stored?

    The kit should be stored at 2 - 8°C upon receipt. Once opened, the plates must be reconstituted immediately. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month.

    How do I request a custom LEGENDScreen™ product with only my specificities of interest?

    For more info, visit:

    What are the guarantees regarding the lyophilized plate compared to the reconstituted plate?

    Lyophilized product has a guaranteed shelf life of 6 months unopened. Reconstituted plates can be used or stored at 2 - 8°C sealed in the dark and used within a month. Be sure to properly seal the plates to prevent evaporation and shield the antibodies from light.

    I have added my own antibody solution to the lyophilized product, will the lyophilized antibody work?

    Yes, as long as the fluorophores on these antibodies are compatible and proper compensation has been applied during acquisition and analysis.

    I am not going to use all the reconstituted antibody solution. Can I keep the left over for later or re-dry the solution in dark?

    The antibody is in a one test per well format. There will not be any antibody left if the full test is used. Customers may decide to use less than the recommended volume per test, but this is not recommended and the performance is not guaranteed. Customers may also selectively transfer certain antibodies from the original plate to a new plate and use after reconstitution. If any antibody is not used after reconstitution, the plate can be sealed and store at 2 - 8°C for a month in the dark. Once reconstituted, re-drying is not recommended as this may result in a loss of signal.

    Can I use my existing institution discount for this new product?


    If I don't have enough cells and use much less than 4 million/mL (~ 3 x 105 cells/well), will it still work?

    This may work with lower numbers of total cells, but we recommend trying to keep higher concentrations of cells for faster analysis. Of course, how many cells are needed depends on the specific application. Successful staining has been done with 1 x 105 cells/well.

    Are these plates made under sterile conditions?

    The plates are not sterile. You would handle them as you would handle most typical flow cytometry staining protocols.

    Can I use half or less of the plate and keep the rest for later?

    Yes. You can use half of the plate or the specificities you are interested in. However, the whole plate should be reconstituted. The half plate of antibodies (or antibodies of interest) must be transferred to another empty plate for the staining. The remaining half must be sealed and stored at 2 - 8°C in the dark and used within a month.

  • Why can’t I fix my cells prior to using Zombie?

    The fixation process can contort and alter the membrane of cells, effectively rendering them as dead. Since the ability of Zombie to stain dead cells is correlated with cell permeability, your results may no longer be a valid representation of dead versus live cells.

    Can I use methanol/ethanol for fixation after using Zombie Dyes?

    Yes, most fixation reagents are fine to be used with Zombie Dyes. However, it should be noted that the dyes can still be sensitive to reactive oxygen species. Light exposure or reagents with hydrogen peroxide can lead to free radical formation, affecting fluorescence.

    How does the performance of your Zombie Dyes compare with competitors?

    Zombie Dyes have been tested against other leading competitors' fixable viability kits and given comparable results. We also highly recommend that you titrate down the amount of each dye used in order to best match the negative signals of your unstained sample and MFI- (mean fluorescence intensity) stained samples.

    Can I use the UV laser to stimulate Zombie Aqua™? If so, can I then use it in conjunction with BV510™?

    While we typically do not test Zombie Aqua™ with the UV laser, its excitation peak suggests it is effectively excited at 355 nm. However, we would not recommend using BV510™ off the violet laser and Zombie Aqua™ off the UV laser at the same time. Due to cross-beam excitation of BV510™ by the UV laser and the violet excitation of Zombie Aqua™, this would lead to significantly increased background and excessive compensation requirements.

    Can I use Zombie and Annexin V to detect apoptotic cells?

    Yes, Zombie can be used with Annexin V to discriminate live, apoptotic, and dead cells. Cells double positive for both Zombie and Annexin V are dead, while Zombie-dim/Annexin V-positive cells are apoptotic. Live cells will be Zombie-low and Annexin V-negative. The advantage to Zombie over PI and 7-AAD is that you can now fix and/or permeabilize the cells to stain for cell surface and intracellular antigens.

    When assessing viability, why is washing not recommended after adding 7-AAD or PI?

    These dyes bind in equilibrium with DNA. Therefore external dye concentration must be maintained during analysis and the dye should not be washed out.

    How is your Annexin made and what sequence does it cover?

    It is made in E. coli, covering human aa Met1-Asp320.

    Is Zombie Aqua™ an equivalent to cell labeling and tracer dyes such as CellTrace™ Violet and CFSE?

    Zombie Aqua™ is not a direct equivalent to such dyes because their mechanism of action and scope of application are entirely different.

    I am concerned about the spillover I am observing from the Zombie dye into its neighboring channels.

    Rule of thumb with Zombie dyes is to titrate them down as much as possible to fit your application. This should potentially help with spillover. Secondly, Zombie positive events represent dead cells and are typically gated out from analysis.

    Can Zombie be used to determine bacteria, yeast viability?

    We have not tested in house bacterial or yeast viability using Zombie dyes. It is not clear whether the difference between surface and intracellular signals will be significantly different in case of non mammalian cells.

    Can I use Zombie with cells suspension containing serum?

    Serum is full of proteins which will sequester the dye and thereby reducing its effective concentration. The basic rule of thumb with zombie is to titrate it based on your specific condition. Titration also helps reduce the background and spillover into other channels.

    Can I use your Zombie dyes for microscopy application?

    Zombie dyes that have been tested for microscopy applications in-house will display data on the product webpage. It should be noted that Zombie may not work for dead cell discrimination in every microscopy application, as a complicated point will be to determine the level of Zombie signal that constitutes a dead cell. Another difficulty may be finding the proper plane for microscopy in order to observe the dead cells.

  • Are downstream applications affected by the magnetic beads bound to the cells?

    We have tested mouse CD4 positive cells that have been isolated from the spleen and lymph nodes with directly conjugated Nanobeads, as well as CX3CR1 expressing cells from mouse bone marrow. The cells appear unaffected as assessed by migration, differentiation and stimulation assays.

    Are there any clone restrictions when checking the purity of isolated cells?

    There are no restrictions for negatively selected cells, as they are untouched by antibodies. For positive selection, there may be clones that are less efficient than others due to possible epitope competition. Please refer to the Technical Data Sheet or contact the Technical Service Group for advice.

    Are your magnetic particles suitable for use with whole blood?

    We have not tested this application yet.

    Can I stain positively sorted cells by flow cytometry?

    Yes, this is possible, but if you are staining for the same marker as used for sorting, we would recommend using a different clone with non-overlapping epitopes.

    Can your magnetic particles be sterile filtered?

    Yes, they can be sterile filtered as the particles are smaller than 0.22 µm.

    Can your magnetic particles withstand freeze/thaw cycles?

    Not recommended, however lyophilized particles can be made available as a custom product.

    Do you offer your base magnetic particles (i.e. without any streptavidin or antibody conjugation)?

    No, not currently.

    Do you provide a custom conjugation service to your magnetic particles?

    Yes, we do. Please contact our Custom Solutions Team at

    Do your magnetic particles get internalized by the cells?

    We have not determined this yet.

    Is there a way to detach your magnetic particles from the cell surface?

    No, not currently.  We have found that cells are functional without the need to detach the magnetic Nanobeads.

    What is the coating on your magnetic particles?

    Hydrophilic polymers.

    What is the composition of your MojoSort™ Buffer?

    The diluted, 1X MojoSort™ buffer contains 1X phosphate buffer saline (PBS) supplemented with 2 mM EDTA and 0.5% BSA. The 5X solution is sterile. To preserve sterility dilute with sterile water under sterile conditions.

    What is the guarantee time of your magnetic particles?

    A minimum of 6 months from the date of purchase.

    What is the shelf-life of your magnetic particles?

    1 to 2 years depending on product type.

    What is the size of your magnetic particles?

    The average diameter is approximately 130 nm.

    What is the storage buffer of your particles?

    The particles are stored in a neutral pH solution containing BSA and sodium azide.

    Will your magnetic particles separate well in other company’s magnetic separation systems?

    It is possible that other magnetic separation systems can be used. To learn more about this please contact our Technical Service Group.

    Are MojoSort™ Nanobeads compatible with other commercially available magnetic separation systems?

    MojoSort™ magnetic particles can be used with other commercially available magnetic separators, both free standing magnets and column-based systems.  Because MojoSort™ protocols are optimized for the MojoSort™ separator, the protocols may need to be adjusted for other systems.  Please contact BioLegend Technical Service for more information and guidance.  We do not recommend using MojoSort™ particles for BD’s IMag™ or Life Technologies’ DynaMag™.

  • What are the advantages of Epitope tags?

    • Tag can be easily and rapidly added to a known gene.
    • Multiple tags can be added if required.
    • Well-characterized antibodies are available.
    • The antibody is specific to the tag, therefore cross-reaction with other proteins is avoided
    • Proteins and protein complexes can be purified using standardized practices.
    • Tagged proteins can be distinguished from otherwise identical untagged proteins
    • Possible to study novel and poorly immunogenic proteins.

    What are the limitations of Epitope tags?

    A few can be as follows
    • A cloned and characterized gene or cDNA must be available
    • The epitope tag may interfere with protein structure or function
    • Epitope-tagged genes can be expressed at abnormal levels due to the use of heterologous promoters
    • The epitope-tagged gene must be introduced into the cell, tissue, or organism of interest.

    How do I introduce the tag?

    The two standard approaches to tagging a cloned gene are: (a) An epitope encoding oligonucleotide is inserted into the coding sequence, or (b) the coding sequence is inserted into an expression vector that already carries the epitope tag. BioLegend does not provide cloning vectors or oligos for cloning. The cloning protocols can be found online or in the literature. When tagging by oligonucleotide insertion, it is important to take into account codon usage preferences for the target cell or organism.

    Will the epitope tag interfere with the function of my protein?

    That has to be determined empirically. However, it is possible that the insertion of the epitope tag may interfere with protein function.

    Where should I put the epitope tag?

    In the literature, the tag has been placed at, or very near, the extreme N or C terminus of the target protein. There are a few reasons behind this choice. Some are historical-like the first proteins to be epitope tagged were tagged at the termini, and when new proteins were tagged it seemed wise to do what had worked in the past. Some of the reasons are practical-tagging is often performed using expression vectors that automatically put the tag at a terminus. And some reasons are theoretical-termini are frequently chosen in the belief that proteins will tolerate additions more readily at these locations than at other sites. While the latter belief may be true-termini are rarely included in active sites, for example-it is also true that many proteins are known for which terminal sequences are critical for function. The termini also appear favorable because they are likely to be on the outside of the folded polypeptide, where one wants the tag to go, and not in the hydrophobic core. But it must be remembered that, due to simple geometry, most of the amino acids in any protein are on the outside, and so, if a protein is tagged at a randomly chosen site, the tag will probably wind up on the outside anyway.

    Why is there no signal with the epitope tag antibodies on a western blot?

    A lack of signal following western blotting may indicate a few different problems.

    1. No or very poor transfer. This can be addressed by quickly checking the membrane with Ponceau-S staining.
    2. The expression level of the GFP-tagged protein may be too low. Load more ug quantity of the sample and include a positive control.
    3. A very diluted antibody may be the problem, try a few different concentrations of the antibody to probe the western blot.
    4. Also, a remote possibility is that the GFP tag is out of frame and is not expressed resulting in a lack of any signal.

    Proteins can be detected with an anti-protein antibody, but not with the epitope tag antibody. What's happening here?

    It is possible the target protein was not tagged, the tag is out of the reading frame, or the protein is degraded.

    How to set up controls for co-immunoprecipitation experiment?

    For negative control, use an unrelated antibody, or control with the same host species, class, and subclass as the immunoprecipitating antibody. For positive control, use an expression vector with only the epitope tag of interest.

    During Western blot with epitope tag antibodies, there are multiple bands.

    Several reasons can account for this:
    1. the early termination of the translation of epitope-tagged protein;
    2. non-specific detection with anti-epitope tag antibody due to no/poor blocking of the immunoblot.
    3. partial degradation of the protein
    4. the concentration of the primary or the secondary antibody is too high.
    5. Washing between the incubations is not efficient

    What applications can I use epitope tagged antibodies for?

    The commonly used applications are western blotting, immunoprecipitation and protein purification. These can also be used for immunofluorescence microscopy and flow cytometry.

  • Will the prices for former NeoClone products remain the same?

    Yes, BioLegend will not re-price these products. But customers can now use their BioLegend discounts and promotions on former NeoClone products.

    Can I still use the former NeoClone catalog numbers to place my order?

    Customers can find products using the former NeoClone catalog numbers on the website, but all products will be re-assigned a six digit BioLegend catalog number, which will be required for ordering.

    Who do I contact about product questions?

    Once the products are available on BioLegend's website, contact BioLegend's technical service team for any product questions.
  • How are BioLegend's carrier-free recombinant proteins shipped?

    Our carrier-free recombinant proteins are shipped on blue ice.  These products have been validated to maintain activity after shipping using blue ice.  

    What is a carrier protein?

    Carrier proteins, such as BSA, improve the stability of the reconstituted protein, and helps prevent the product from sticking to the wall of the vial.

    What is the difference between the carrier-free and the non carrier-free recombinant proteins?

    All our carrier-free and animal-free formats of recombinant proteins do not have any additional carrier proteins such as BSA in the formulation. Typically our ELISA standard recombinants have carrier proteins added to the formulation for added stability and to avoid the product from sticking to the wall of the vial. When the presence of carrier is not desirable (e.g., in-vivo applications), carrier-free proteins can be used directly. When carrier proteins do not affect the outcome in a study, the customer can decide what type of carrier protein they would like to use and whether it is necessary to add it to their stock.

    Can I use different recombinant proteins from different companies for my ELISA?

    Antibodies used are different in different kits. The specificity of the antibodies partially dictate how much signal is being detected. Recombinant standards used are different. First of all, different kits may use recombinant proteins expressed and purified using different method. Second, recombinant proteins expressed from E. coli from the same source can show greater than 10 fold difference in term of immunoreactivity from lot to lot, primarily due to refolding inconsistency. Third, different kit standards can be produced and calibrated against different references. So far there is no universally accepted standardization for cytokine immunoreactivities. Each BioLgend ELISA product was developed and validated with reagent concentrations and protocols optimized for best analytical robustness. Any changes to the reagents (standards, antibodies, matching matrices) and protocols etc all affect the final assay performance.

    How does the activity of your recombinant proteins compare to competitors?

    We quality control each and every lot of recombinant protein. Not only do we check its bioactivity, but we also compare it against other commercially available recombinant proteins. We make sure each recombinant protein’s activity is at least as good as or better than the competition’s. In order to provide you with the best possible product, we ensure that our testing process is rigorous and thorough. If you’re curious and eager to make the switch to BioLegend recombinants, contact your sales representative today!

    What is the specific activity or ED50 of my recombinant protein?

    The specific activity range of the protein is indicated on the product datasheets. Because the exact activity values on a per unit basis can largely fluctuate depending on a number of factors, including the nature of the assay, cell density, age of cells/passage number, culture media used, and end user technique, the specific activity is best defined as a range and we guarantee the specific activity of all our lots will be within the range indicated on the datasheet.

    What should I reconstitute the protein with? What do you recommend for its long-term storage?

    Most of our carrier-free recombinants are shipped in liquid form, so there is no need for reconstitution. Our animal-free recombinant proteins are shipped in lyophilized form and protein reconstitution information is indicated on the respective datasheets. If you need to make dilutions, refer to the formulation on the product data sheet. Stock solutions should be prepared at 50-100 μg/mL in buffer containing carrier protein such as 1% BSA or HSA or 10% FBS (for chemokines, use either BSA or HSA). For long-term storage, aliquot into polypropylene vials and store in a manual defrost freezer. Avoid repeated freeze/thaw cycles.

    For reconstitution of our lyophilized recombinant proteins (ELISA Std, animal-free, and some carrier-free) please refer to the Certificate of Analysis and/or Technical Datasheet included with the product and follow the instructions. 

    Are BioLegend’s recombinant proteins suitable for in vitro and in vivo bioassays?

    BioLegend’s recombinant protein solutions are 0.2 μm-filtered prior to bottling by membrane filtration method. In addition, our recombinants are endotoxin-tested and are guaranteed to have levels less than 0.1 ng per μg protein. All our recombinants can be safely used for both in vitro and in vivo bioassays.

    Do you test the bioactivity of your recombinant proteins with in vivo assays?

    We typically validate the activity of the proteins with in-vitro assays as described on the data sheet and not with in-vivo testing. However, all our carrier-free and animal-free formats can be used for in vivo applications as is evident from many customers who have successfully used it for this purpose.

    Does specific activity of a recombinant protein vary between lots?

    Specific activity will vary for each lot and for the type of experiment that is done to validate it, but all passed lots will have activity within the established ED50 range for the product and we guarantee that our products will have lot-to-lot consistency. Please conduct an experiment-specific validation to find the optimal ED50 for your system.

    What is the difference between laboratory (observed) units and international units?

    There is no direct relationship between International Units and the units that are calculated using the inverse of the specific activity because we do not use the International Standard provided by WHO (National Institute for Biological Standards and control). The best way to compare the activity of two sources of recombinants is by doing the bioassay side by side using the same system.

    How do you convert activity as an ED50 in ng/ml to a specific activity in Units/mg?

    Use the formula Specific activity (Units/mg) = 106/ ED50 (ng/mL)

    Have your recombinants been tested for stability?

    Our testing shows that the recombinant proteins are able to withstand room temperature for a week without losing activity. In addition the recombinant proteins were also found to withstand four cycles of freeze and thaw without losing activity.

    What is the difference between carrier-free and animal-free categories of recombinant proteins?

    Our animal-free products are proteins that go through the entire production process without touching any animal containing components. This includes using animal-free media and purification equipment that are animal component-free. Some studies which are particularly sensitive to contamination by mammalian pathogens may require the use of animal-free products. Our carrier-free products do not contain any carrier protein in the solution, as expected, but they are produced using animal-containing components. Both versions are expected to have similar activity and function, though specific activity is lot-dependent.

  • What are possible causes of High Background in my Western Blot?

    * Transfer buffers may have become contaminated.
    * Post-antibody washes may not have been performed with sufficient time or volume.
    * Blocking and incubation agents were not freshly prepared or were too dilute.

    What are possible causes of No Signal/Poor Signal in my Western Blot?

    *  Transfer efficiency may have been poor. Check protein transfer by staining the gel and/or membrane.
    * Incorrect storage of antibodies or ECL western blotting detection reagents.
    * Insufficient protein may have been loaded on the gel. Depending on the location of the target protein, membrane or nuclear preparations may be required (instead of whole cell lysates).
    * Film exposure time may have been too short.

    Why it is hard for flow cytometry or ELISA compatible antibodies to work for western blot?

    The antibodies that are tested via flow cytometry or ELISA work by recognizing 3D target conformation and epitopes. Harsh treatment associated with WB such as boiling leads to linearization of the target epitopes, which may then lead to non recognition of the target.

    If you have not tested/validated the antibody for WB, why do you claim it works for WB?

    We try to provide additional information such as published literature about a particular antibody clone's compatibility with other reported applications so that end users can make an informed decision before embarking on an experiment. It is recommended to consult additional literature to get further details about the protocol and/or particular cell type or experimental situation. Each experiment is unique and so tissue specific validations and titrations are necessary.

    What is your protocol for treatment of your sample prior to generating the web data?

    Please contact technical service for details.

    Do you offer blocking peptides, positive control reagents and cell lines?

    We carry a Posi-Tag Epitope Tag Protein that can serve as a positive control for Western Blotting with epitope tags, but otherwise, please try

    Why is my actual band size different than the predicted molecular weight?

    The size of the target protein can be affected by many factors such as:

    • •Post-translational modification such as phosphorylation, glycosylation can increase the size of the protein.
    • •Post-translation cleavage - e.g. some proteins may get cleaved to the active form from pro-protein form via post translational cleavage.
    • •Different sized proteins produced from the same gene can be produced by alternative splicing (Splice variants).
    • •Overall charge on the protein based on amino acid composition can also affect the protein migration in a gel.
    • •Strong protein-protein interactions can potentially lead to Multimers thereby leading to the appearance of higher size bands. This is usually prevented in reducing conditions and boiling of the sample before loading to the gel.

    How much antibody should I use or what titration range you recommend for WB?

    0.5 μg/ml to 4 μg/ml range can be used for titrations.

  • Can antibody X be used for immunohistochemistry? What concentration do I use?

    Typical concentrations of monoclonal antibodies for use in IHC are from 5-25 µg/ml. Polyclonal antibodies can be used at a range of 1-10 µg/ml. Some products are quality tested in-house for IHC applications, while others will indicate on the datasheet if an antibody has been published for use in this application. In addition, you can do a lit search with the clone name and immunohistochemistry/paraffin/frozen to see what the protocol details are.

    Do I need to perform antigen retrieval on my formalin-fixed, paraffin-embedded samples prior to staining?

    In most cases, this is true.  Antigen retrieval helps both the accessibility of the antibody to the tissue and also counteracts the fixation effects on the recognized epitopes. Check the application references for any additional details for IHC or IF experiments.

    Can I use GFP expressing cells with Alexa Fluor® 488 or FITC?

    No, due to spectral emission overlap you will not be able to distinguish between GFP and Alexa Fluor® 488 or FITC.

    You say that the antibody is reported for IHC and when I read your reference it didn't provide me with details like how much antibody to use or detailed protocol.

    If an antibody is listed as being quality-tested or validated for IHC, we can look up the protocols for you. If this application is reported in literature for IHC, we have not specifically tested the product for this application. It relies on information from publications. Of course, we seek to provide the most updated information on these publications, so we can update our datasheets if the information is incorrect.

    Can I use antifade my live cell imaging microscopy?

    Antifade should not be added for live cell imaging as it will deprive the cells for oxygen.

    Are fluors such as BV570™, BV605™, BV650™, BV711™, BV785™, PE/Cy7, PE/Cy5, APC/Cy7, PE, APC, and PerCP useful for immunofluorescence?

    These dyes due to their vulnerability to photo bleaching these are not recommended for IF. Alexa Fluor®, Dylight, and BV421™ fluors are good alternatives for immunofluorescence.

  • Which markers can be stained?

    160+ known markers have been verified. To see a list of verified markers, please go to:

    Are these cells viable?

    No they are not.

    Can the cells be used for T or B cell sorting using magnetic bead isolation?

    Yes, it should be possible, although this has not been verified by BioLegend.

    Can they be used for functional assays?

    This will not work, since the cells are not viable.

    Can you freeze the cells after reconstitution?

    We do not recommend freezing the cells after reconstitution.

    Can they be used to detect intracellular cytokines such as TNF-α or IFNγ?

    Veri-Cells™ PBMC and Veri-Cells™ CD4-Low PBMC will not have detectable TNF-α or IFNγ. Please inquire about a custom made Veri-Cells product at

    Can the cells be activated after rehydration?

    No, they cannot be activated.

    Can a constant percentage of each population be expected in each lot?

    Yes, within each lot, cell populations remain constant.

    Is RNA from Veri-Cells™ able to be used for analysis, for example RT-PCR or RNA flow?

    We have not tried this yet, but it may be possible.

    Can the buffer included with the cells be used to keep thawed PBMCs alive?

    We have not tried this.

    What is the expected percentage of CD3+ cells?

    This information is provided in the COA for each lot, it would be similar to what you would see in the “normal” population.

    Are these cells virus/pathogen free?

    These are tested to be free of HIV, HBV, syphilis, and HCV.

    How do we QC these cells?

    We QC for T, B, NK markers for Veri-Cells™ PBMC products - CD3, CD19, CD4, CD8, CD56/CD16.

    Do Veri-Cells™ stain 100% positive for 7-AAD/DRAQ7™?


    Are there any RBCs in Veri-cells™?

    There may be a few, but the majority of RBCs are not present in Veri-Cells™ preparations.

    Can I treat this like any other sample of PBMCs?

    Yes, make sure to use the Reconstitution buffer for the staining.

    Is washing required after staining?

    Yes, it is recommended to wash after staining. We recommend using BioLegend's Cell Staining buffer (Cat. No. 420201) for the wash step.

    How much antibody should I use to stain Veri-Cells™

    Depending on application pursued it may require diluting the antibodies prior to use.

    Are the cells fixed?

    They are treated with our proprietary mixture prior to lyophilization.

  • Why do I get a high background when my enzyme definitely has no free Pi?

    This is almost certainly caused by inadequate mixing of the Stabilizer. This results in a high background signal because of non-enzymatic decay of ATP substrate. The Stabilizer is added in a relatively small volume (20μL), and the operation of pipetting up and down with a pipette set to 20μL volume may not result in sufficient mixing when the total volume is 270μL. Try pipetting up and down while stirring at the same time. Alternatively, add the Stabilizer with one pipette set at 20μL volume and mix using a larger pipette set to ~150μL volume. This ensures thorough mixing of the Stabilizer solution with minimal effort.

    I have 5% DMSO in my assay. Can I use PhosChrome™?

    Yes, the reagent is designed for drug screening work and other situations require DMSO.

    I have phosphate in my enzyme. What can I do?

    You can dialyze or desalt the enzyme into a phosphate-free buffer.

  • What are MHC tetramers and what can you do with them?

    The T-cell mediated innate immune response is defined by the interaction between antigen presenting cells and T cells, through the Major Histocompatibility Complex (MHC) and the T cell receptor (TCR). MHC molecules present a peptide to antigen-specific T cells that recognize this peptide. Soluble, monomeric MHC molecules bind very weakly to the TCR. However, by making a tetramer through a fluorescently labeled streptavidin conjugate, the complex binds to several TCRs, creating a more stable interaction and making it useful for flow cytometric detection of antigen specific T cells.

    What is Flex-T™?

    Flex-T™ (Flexible-Tetramers) is BioLegend’s brand name for our Soluble MHC product line. It encompasses monomers, the ultraviolet (UV) peptide exchange technology, and all associated products and applications.

    Is there any advantage in buying biotinylated monomers?

    Yes, the same monomer can be assembled with different Streptavidin conjugates, providing great flexibility for color choices.

    Additionally, it can be stored frozen and the tetramer assembled shortly before the experiment. This increases the storage time of the reagent.

    What is the peptide exchange technology and what’s the advantage of using it?

    Flex-T™ MHC monomers are loaded with a peptide that can be degraded by the use of a UV light source. This allows for a peptide exchange when the UV irradiation is done in the presence of the peptide of interest (which is not UV-labile). This flexibility permits the screening of virtually any peptide of interest with enough affinity for the MHC allele that it is loaded onto.

    What are the specifications of the UV source?

    Long-wave UV, 366 nm, 8 Watts (We recommend, for example, CAMAG cat# 022.9115, or Ultraviolet Crosslinker CL-1000). The distance from the solution to the light source should be 2 – 5 cm (approximately 0.8 – 3 inches).

    How do I evaluate the efficiency of the peptide exchange?

    Follow the protocol for HLA class I ELISA. An assay positive control can be purchased separately (Cat#280301), and it can be diluted to a high, medium, and low concentration. Signal intensity can be correlated to affinity of the peptide.

    Is there any peptide length recommended?

    There are no special requirements for the peptides. Peptides that naturally bind to MHC molecules will bind to Flex-T™ reagents. For class I molecules, typical length is about 8 – 10 amino acids. Class II molecules accommodate longer peptides, about 14 – 20 amino acids1.

    1) Mohan JF and Unanue ER. Unconventional recognition of peptides by T cells and the implications for autoimmunity. Nat Rev Immunol. 2012 Oct;12(10):721-8.

    Do I need to know the sequence of the UV-labile peptide?

    The sequence of the UV-labile peptide is not needed to use the reagent. MHC molecules are not stable without a peptide, so these peptides are used just for two purposes: stabilize the MHC molecules and serve as a place holder to be substituted by the peptide of interest.

    If I am still interested in “fixed” peptides, what do you recommend?

    Please see the linked document titled “Peptide Sequences”. The document contains a table with commonly used peptides by allele to study antigen specific T cells.

    I am interested in finding novel peptides instead, are there any resources for this?

    There are a number of databases and webpages that can help, these are three of them:

    Is it feasible screening not just peptides, but also several specificities of antigen specific T cells in one sample?

    With the traditional pre-assembled Tetramer approach, this is difficult to do, and not cost-effective. With Flex-T™ technology, as there is more flexibility to assemble the tetramers, it is easy and affordable to screen a sample for several specificities2. To facilitate this approach, a combinatorial color coding system has been developed. Please visit Flex-T™ webpage at, Combinatorial Color Coding tab for a detailed description about how to do this.

    2) Hadrup SR et al. Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers. Nat Methods. 2009 Jul;6(7):520-6.

    Do you offer mouse monomers or tetramers?

    Not currently.

    Do you offer custom products and services?

    Yes, please contact our Custom Solution Team with your request at, or contact your local BioLegend representative.

  • How do I know if my chromatin has been sheared?

    Purify the DNA from your sheared chromatin samples and run the DNA on a 1-2% agarose gel. DNA should be sheared to a range of 100-900 bp. Below is an example of chromatin from a number of different cells lines that has been sheared optimally using enzymatic digestion compared to chromatin which was over-digested.

    Lane 1: Ladder, Lane 2: HeLa, Lane 3: Jurkat, Lane 4: NCCIT, Lane 5: HeLa IL-6Lane 1: HT1080+ IFNγ, Lane 2: NIH353, Lane 3: HT29, Lane 4: HCT116, Lane 5: Ladder

    Which points in the protocol can I stop and save my experiment?

    Sheared chromatin may be saved at -80°C prior to performing the immunoprecipitation. If you are saving the chromatin, we would recommend aliquoting the chromatin as it can be sensitive to freeze thaw cycles. Alternatively, purified DNA can be saved prior to downstream analysis.

    Should I use sonciation or enzymatic shearing?

    Sonication may be ideal for difficult to lyse cell types and provides random fragmentation but may damage epitopes. It is not suitable for Native ChIP experiments (in which the chromatin has not been cross-linked). Enzymatic digestion is milder and can be used in Native ChIP experiments but will exhibit sequence bias and may not be suitable for hard to lyse cells.

    What cell types have been validated with the Go-ChIP-Grade™ kit?

    Human and mouse cell lines such as HeLa, Jurkat, Daudi, THP-1, NIH3T3, and 293T have been validated using this protocol. Other cell types (plant, yeast, tissue, FFPE samples, etc.) may require optimization.

    How do I determine optimal number of cells for ChIP assay?

    An important consideration when performing ChIP is the amount of chromatin that will need to be loaded to the column in order to elute sufficient IP’ed DNA for downstream analysis. Sufficient cell numbers should be used so that at least 3µg of chromatin can be used per IP. Start with 1-15 million cells. However, the cell number can be scaled up and the reagents need to be adjusted accordingly.

    How much antibody for target protein should be used per ChIP?

    This should be determined empirically by the end-user. Refer to the datasheet for our Go-ChIP-Grade™ Purified Antibodies, or other ChIP-validated antibodies. The suggested dilution of the Go-ChIP-Grade™ anti-RNA Polymerase II Antibody; Clone 8WG16 (positive control) is 1:300 – 1:500 by volume.

    What is causing high background?

    The quality of the ChIP antibody has a major impact on the success of the assay. Use only ChIP-validated antibodies. Make sure to have good quality chromatin samples with fragments between 150-900bp. Insufficient DNA shearing leads to longer DNA fragment length that can cause high background in downstream experiments. Insufficient wash steps can also leave traces of non-specific chromatin alongside enriched DNA. If background remains high, include an additional wash step during the IP protocol.

    Why do I not have any enrichment?

    Make sure to only use ChIP-validated antibodies and follow the instructions for the amount of antibody to be used or titrate the antibody for your experiment. It is also recommended to include ChIP validated positive and negative controls (included in the kit) to validate the efficiency of your ChIP assay. Good quality chromatin samples and chromatin fragment sizes between 150 – 900 bp are also critical. Other factors can include whether cells were effectively lysed, over-fixation and reduced antibody binding due to decreased epitope availability, and insufficient starting material (chromatin sample per IP).

  • What is TotalSeq™ and how do they work with established workflows (CITE-seq, and REAP-seq)?

    TotalSeq™ is BioLegend’s brand of antibody-oligonucleotide conjugates, to enable simultaneous analysis of proteins and mRNA in single cells. CITE-seq and REAP-seq are two similar workflows to do simultaneous protein and mRNA analysis, and the TotalSeq™ conjugates integrate seamlessly into these workflows.

    What are “Hashtags”?

    Hashtag reagents are intended to be used for sample barcoding, which allows users to combine multiple samples into a single lane, then de-multiplex during analysis. Instead of select antigen-specific antibodies, the hashtags are designed so that they are specific for human or mouse cells, and to cover as many cells as possible. For human samples, the hashtags are made of two antibodies that recognize ubiquitous surface markers, CD298 and β2 microglobulin, each conjugated to the same oligonucleotide containing the barcode sequence. For mouse samples, the surface markers are CD45 and H-2 MHC class I. The conjugates are already pre-mixed and ready to use.

    What’s the benefit of Cell hashing?

    Hashing allows customers to run multiple samples in a single lane of a 10x Genomics Chromium instrument, or equivalent, which increases the number of cells or samples that can be analysed simultaneously, significantly decreasing the cost of the experiment.

    Does co-staining for FACS and TotalSeq™ interfere with the workflow?

    We have not tested this, but there is no reason to believe that non-competing clones for the same target would be problematic. Similarly, a sub-saturating concentration of both reagents against the same target should be tolerated by the cell/technique. The original CITE-seq paper by Stoeckius et al.(Fig. 2) demonstrated that cells can be co-stained for downstream analyses. Although the authors did not use current TotalSeq™ reagents, the technology is the same. In addition, other CITE-Seq users have also demonstrated this approach. Please contact our technical service group for more information.

    Is it possible to do FACS sorting first then do CITE-Seq, while performing both antibody-oligo and antibody-fluorophore labelling at the same time? Will the oligo conjugate withstand sorting?

    The oligo should withstand sorting. However, based on theoretical considerations we recommend to use non-competing clones, to minimize impact in either FACS or sequencing signal/reading. Even if exposed for a very short time, we have not evaluated the effect of ultraviolet or violet lasers on the oligo conjugates. An alternative to flow sorting could be magnetic bead enrichment, such as our MojoSort™ platform.

    How many TotalSeq™ antibodies is it possible to multiplex?

    A published article multiplexed 82 antibodies conjugated to oligonucleotides. The authors did not use current TotalSeq™ antibodies, but the method is very similar, which they termed REAP-seq. There is no reason to believe that larger panels are not possible.

    How many cells do you need per experiment?

    This depends on the biological question that needs to be answered. From the technical perspective, when using 10X Genomics Chromium, it is recommended to load 5,000 – 10,000 cells per lane. The use of Hashtags can increase that number. For ease of staining, we typically recommend 1 million cells, but this number also depends on availability of cells and the operator’s ability to handle low cell numbers.

    What is the baseline copy number for detection of protein expression (via TotalSeq™ labelling)?

    This has to do mostly with antibody affinity, titration, and level of antigen expression. BioLegend and some collaborators are working on titration data. There is also some data already published. In theory, as long as the antibody can bind one molecule, PCR amplification and sequencing should pick it up, but there is always background noise. A common way to control for this is to add negative cells to your experiment. For example, if working with human PBMCs, use mouse cells and vice versa.

    Is autofluorescence an issue as in flow cytometry?

    No, sequencing as the final readout is not affected by cell autofluorescence.

    Can CITE-seq be extended to micro-RNAs?

    This is a technically challenging application as micro-RNAs are very small, and in many cases will be as small as or even smaller than the required primers to execute the protocol. It is not possible at the moment.

    Is it possible to run CyTOF® and TotalSeq™ (CITE-seq) alongside each other?

    Not in the same experiment; it is not possible because CyTOF® requires its own instrument and protocol, and it destroys the sample in the process. To do so, it is necessary to split the sample and run both CyTOF® and CITE-seq assays. Note that CITE-seq generates equivalent data for surface proteins as CyTOF® with higher panel multiplexing capabilities and at a single-cell level.

    How is the oligo tag bound to the antibody so that it does not interfere with antibody binding to target protein?

    The oligo is attached to the antibody in the same method as some of our fluorophore-conjugated antibodies that are quality tested for flow cytometry. Once the antibodies have been conjugated to the oligonucleotide, we verify by flow cytometry that the antibody can still bind to its target. This is part of our quality control process for TotalSeq™ conjugates.

    Is it necessary to sort samples prior to CITE-seq? What should I consider when deciding to pre-sort, or not?

    If the populations of interest can be clustered/identified without sorting, the likelihood that you need to pre-sort is minimal. However, if the cell number in the cluster is very small, it may not be sufficient to draw conclusions when compared to control, or other cell populations. In which case, enrichment of this population prior to CITE-seq analysis may help with obtaining more meaningful sequencing data that is selective for your rare cell population(s) of interest. We also recommend to sort out dead cells if the viability in a sample is lower than 95%.

    Why is it essential to remove dead cells prior to subjecting samples for CITE-seq?

    Running dead cells during CITE-seq essentially leads to “wasting” single cell runs on dead cells, which may ultimately lead to sub-optimal data, or not processing enough cells to pick up the positive events, especially if the frequency of your target cell is very low. Dead cells can also be removed using magnetic particles. If performing CITE-seq before eliminating dead cells is required, it is possible to use bioinformatics methods to try to clean up low quality events (which may include dead cells). This could be a more complex approach, but in such cases, we recommend the following reading:

    How can I titrate TotalSeq™ antibodies?

    We are still in the process of identifying optimal concentrations for the use of TotalSeq™ reagents. However, here is what we can recommend for titrations:

    1. Performing titrations using the actual CITE-seq method is costly, but using Hashtags can make the process more affordable. See Fig. 3 and associated methods of this publication for more information.
    2. Perform flow cytometry experiments to find the optimal antibody amount, using the same clone conjugated to PE. The flow cytometry antibody amount translates well to the CITE-seq amount.
    3. Label the cells with different concentrations of TotalSeq™ antibody, and then use a poly-dT oligo conjugated to a fluorophore, such as Alexa Fluor® 647, as a secondary reagent to detect the TotalSeq™ antibody. This is a more direct method as compared to the ones listed above, but it requires the use of the actual TotalSeq™ antibody.

    Why should I generate ADT/Hashtags libraries separate from mRNA?

    To provide flexibility when sequencing. Libraries can be mixed at different ratios before sequencing depending on the number of reads needed.

    I need to order primer; where do I order them and what purity is needed?

    There are several companies offering primer synthesis. We can recommend IDT technologies. For additive primers, desalt purification is sufficient. For index primers, either HPLC or PAGE is needed.

    What tools are available for data analysis?

    Cell Ranger 3.0 can be used for barcode count, although some reagents or applications may not be supported by 10x Genomics. Useful resources include CITE-Seq-Count, and the Satija Lab. Please contact our technical service group ( for further information.

  • What is the concentration of the 1000X Monensin Solution (420701)?

    Monensin is provided at a 2mM solution.

    What is the concentration of the 1000X Brefeldin A Solution (420601)?

    Brefeldin A is provided at a 5mg/ml concentration.

    Why do I need to use Annexin V Binding Buffer?

    Annexin V binding requires the presence of calcium in the solution.  So, we provide Annexin V Binding Buffer (cat # 422201), which is optimized for the best performance of Annexin V staining.

    How long can I store an antibody after I have diluted it?

    We would not recommend extended storage of the product after dilution.  It is best to prepare the dilution as needed on the day of use.  Because there are no preservatives in these buffers and they could be susceptible to changes in pH, long term storage greater than a day or two is not recommended.

    The Brefeldin A solution freezes at 4°C . Is the solution still OK?

    We recommend to thaw in warm water after removing from the fridge.  The Brefeldin A is dissolved in DMSO, which freezes at 18°C, so this is normal.  No one has reported any problems with the freeze/thaw cycles and this is how we recommend it is stored.   If customers are concerned, they can aliquot the Brefeldin A to avoid the freeze thaw.

    Can I use my other company's buffers with your antibodies?

    How do I choose monensin or Brefeldin A solution?

    Generally, Brefeldin A is more toxic for longer term incubations, so for shorter stimulations (6 hrs or less) use Brefeldin A.  For longer stimulations use monensin.  We recommend optimization for each cell type and protocol.

    When do I use FluoroFix™?

    FluoroFix™ (cat# 422101) is designed for gentler fixation of cells stained with fluorescent labeled antibodies, including tandem dyes.

    Can I keep my stained cells in your FluoroFix™ Buffer (422101) for several days before acquisition?

    Keeping stained cells in any fixative for longer periods of time is not recommended. You can use FluoroFix™ Buffer as recommended on the data sheet, followed by washing with cell staining buffer and then at the last step, resuspend your fluorofixed cells in cell staining buffer till you acquire your samples in a day or two.

    Is your Foxp3 buffer set compatible with anti mouse and/or human cytokine antibodies?

    Nuclear permeabilization generally creates larger pores in a cell that can allow cytokines to leak out. In order to find out whether this occurs, you would have to run a sample fixed and stained with a non-nuclear permeabilizing buffer (such as Cat. No. 421002).

    Can your Nuclear Factor Fixation and Permeabilization Buffer Set (cat# 422601) and FOXP3 Fix/Perm Buffer Set (cat# 421403) be used in place of each other?

    You have to try it in your system but it should work.
Forgot your password? Reset Password
Request an Account