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    Technical frequently asked questions (FAQs)

    Frequently asked technical questions, answered by our Scientific Support team.

    Can I get a free sample of a product?

    We do not offer free or trial sized samples for testing purposes. Our policy is that if an antibody does not work as specified on the datasheet, we will offer a replacement or refund.

    If the antibody is being used in an untested species or application, we cannot offer a replacement or refund; however, we invite you to use our Abreviews system to let us and other scientists know how the antibody performed. We publish all Abreviews, both positive and negative, on the online datasheets of each product.

    The Abreviews system allows researchers to submit reviews of our products in exchange for Abpoints. Abpoints can be redeemed for discounts on future antibody purchases or rewards such as Amazon gift certificates. You can submit Abreviews on the product datasheet.

    Learn more information about Abreviews

    Can I get a discount?

    We do not offer discounts for one vial of product. We can arrange discounts on orders for five vials or more. This can be five vials of the same product, or mixed products. If you would like to place a bulk order, please contact our customer services team for further information.

    Is the information on the datasheet up-to-date and correct?

    The datasheets contain the most up-to-date information we have available to us about this product. Where we find additional information, the datasheets are updated live on the website immediately, so they should never be out of date.

    How should I choose a suitable secondary antibody?

    Secondary antibodies should be raised against the host species as the primary antibody you are using. For example, if your primary is a mouse monoclonal, you will require an anti-mouse secondary. We recommend you check the datasheet of the secondary antibody to ensure it is tested in the application you will be using. We provide a wide range of secondary antibodies conjugated to a range of fluorochromes and chromogens.

    A couple more tips:

    • To find your own secondary antibody, choose secondary antibody from the 'Products' section of the navigation menu at the top of all pages. Select the host species of your primary antibody. You will then have a list of different conjugates to choose from. Select the appropriate conjugate and then view the datasheets on the list of suitable antibodies provided.

    View our more detailed page on How to choose a secondary antibody.

    How should I choose an isotype control?

    Isotype controls are used to confirm that the primary antibody binding is specific and not a results of non-specific Fc receptor binding or other protein interactions. The isotype control antibody should match the primary antibody’s host species, isotype, and conjugation. For example, if the primary antibody is a FITC-conjugated mouse IgG1, then you will need to choose a FITC-conjugated mouse IgG1 isotype control.

    To find a suitable isotype control on our website, go to the 'Products' section of the top navigation menu. Choose "Isotype control", then the species and subclass of your primary antibody. This will give you a range of isotype controls for use with that species and isotype of primary antibody.

    Isotype controls for polyclonals:
    Most isotype controls are monoclonal. They are not suitable for use with polyclonal antibodies as these contain more than one IgG subclass. However, we do provide several polyclonal isotype controls for use in these circumstances.

    How should I choose a positive control?

    If you require a suitable positive control, please check the datasheet, which will often have a suggested positive control. Always ensure the tissue or cell line you use is from a tested species. Not all the datasheets will have a suggested suitable control, and we recommend the following in these circumstances:

    • Check to see if there are any Abreviews for the antibody. Any tissues, cells or lysates that have been used successfully by these customers can be considered a suitable positive control.
    • Try looking at the Swiss-Prot or Omnigene database links on the datasheet. These databases will often have a list of tissues that the protein is expressed in. These can also be considered suitable positive controls.
    • Check the GeneCards entry for the protein. This will usually provide you with relative levels of expression in various tissues.
    • If you still have difficulty finding a suitable control, we recommend doing a quick literature search on PubMed to see which tissues and cells express the protein of interest.

    What antigen retrieval method should I use with this antibody?

    We do not always have information available on the most suitable antigen retrieval method for an antibody in immunohistochemistry. This will often require a certain amount of optimization by the end user.

    Visit our IHC protocols page for more information on antigen retrieval methods.

    What concentration of primary antibody should I use?

    For antibodies where we state “Use at an assay dependent dilution”, it can be a bit daunting to find a starting point.  The chart below gives some starting dilutions to use based on the purity of the product and the application you will be using the antibody in.

    Unpurified antibodies will not have a concentration stated on the datasheet. For most whole antiserum, culture supernatant or ascites fluid products the concentration has not been determined. Unpurified antibody preparations vary significantly in specific antibody concentration.

    If the specific antibody concentration of a given unpurified antibody preparation is unknown, one may refer to the “concentration estimate” area below as a guideline for estimation.

    Please remember that these dilutions and concentration estimates are simply recommended as a starting point, and it may be necessary to adjust the dilution based on the experimental results.

    Tissue culture supernatantAscitesWhole antiserumPurified antibody
    WB/dot blot1/1001/10001/5001 ug/ml
    IHC/ICCneat - 1/101/1001/50 - 1/1005 ug/ml
    EIA/ELISA1/10001/100001/5000.1 ug/ml
    FACS/Flow cytometry1/1001/10001/5001 ug/ml
    1/1001/50 -1/1001 - 10 ug/ml
    Concentration estimate1 - 3 mg/ml5 - 10 mg/ml1 - 10 mg/ml

    How should I store my antibody?

    We recommend always storing the antibody as directed on the datasheet. We are unable to guarantee how the antibody will perform if not stored as stated on the datasheet.

    Read our antibody storage guide

    How should I aliquot my antibody?

    The size of the aliquots will depend on how much one typically uses in an experiment. Aliquots should be no smaller than 10 μl; the smaller the aliquot, the more the stock concentration is affected by evaporation and adsorption of the antibody onto the surface of the storage vial.

    Read our antibody storage guide

    Why was this antibody discontinued? Can I get more of this antibody?

    We sometimes have to discontinue a product. This can be for several reasons, such as poor sales, unavailability from the originator, or production difficulties. On some occasions, this can be for quality issues, but this is not always the case.

    We understand the inconvenience this causes customers, particularly if it is an antibody they have been using for some time. If we have a suggestion of a replacement for the antibody, we will supply the catalog number when you attempt to bring up the discontinued product’s datasheet .

    Always check to ensure the alternative antibody is suitable for your application and species. If not, please search our catalog for another suitable alternative.

    Any antibody that is listed as unavailable on our website has had its stock exhausted, and we cannot obtain any more of the product.

    Are there any publications using this antibody?

    Our datasheets are updated as soon as we receive new information. All publications that we are aware of using a particular antibody will be listed on the "References" tab of the antibody datasheet.

    Is the antibody tested in another species / application?

    All the species and applications we are aware of that an antibody has been tested in will be stated on the datasheet. These are updated as soon as we receive any new information.

    If you decide to go ahead and purchase a product for use in an untested application of species, we encourage you to submit an Abreview via the online product datasheet. We always encourage customers to send their results back to us, whether positive or negative, and we make all product information available to researchers. We reward each published Abreview with Abpoints, which can be used for discounts off future purchases or other rewards.

    More information about Abreviews

    How can I determine whether an antibody may detect in an untested species?

    Abcam are unable to guarantee that an antibody will work in an untested species, even if the sequence alignment is high. There are many variables involved in the determining whether an antibody will bind in another species.

    If there are no alternatives available, and it is necessary for you to consider purchasing an antibody for use in a species that is not tested, we recommend checking the sequence alignment of the immunogen with the protein you are interested in. Sequences can be found on the Swiss-Prot protein database link on the online datasheet. There are also websites that provide a tool for calculating the percentage alignment.

    Visit EMBL-EBI website

    Take a copy of the immunogen sequence of the antibody and align with the protein sequence from the species you would like to test.  We recommend an alignment score of over 85% as a good indication that an antibody may cross-react. Please note that even if over 85%, we are still not able to guarantee how well the antibody will perform.

    Will this antibody cross react with another isoform of this protein, or protein from the same ‘family’?

    For some of our antibodies cross reactivity data is available. Please check the ‘specificity’ section on the datasheet. If information is not available here, we recommend checking the sequence alignment of the immunogen with the isoforms or other proteins you are interested in.& There are several websites that provide a tool for calculating the percentage alignment.

    Visit EMBL-EBI website

    Take a copy of the immunogen sequence of the antibody and align with the protein sequence from the species you would like to test. We recommend an alignment score of over 85% as a good indication that an antibody may cross-react. We would therefore recommend that the percentage alignment should be much lower that this to indicate there should be no cross reactivity.

    Are you able to provide the immunogen sequence for this antibody?

    For some of our antibodies the immunogen sequence is not readily available on the datasheet. We invest a lot of time and effort into selecting immunogen sequences in order to deliver the most effective antibodies. We sometimes decide to protect this information by giving this information on request to individual customers only. This will be noted on the datasheet by the following link ‘Read Abcam's proprietary immunogen policy’. If you require the sequence for your experiments then please do contact our Scientific Suppport team.  For some externally sourced products, the immunogen sequence is proprietary information and we are unable to release this information, even on request.

    Please note that the specific epitope (the antibody binding region within the immunogen) will very rarely have been mapped for commercially available antibodies.

    Why is the actual Western blot band size different from the predicted?

    Western blotting is a technique that separates proteins based on size. In general, the smaller the protein the faster it migrates through the gel. However, migration is also affected by other factors, so the actual band size observed may differ from that predicted. Common factors include:

    • Post-translational modification - e.g. phosphorylation, glycosylation etc, which increases the size of the protein
    • Post-translation cleavage - e.g. many proteins are synthesized as pro-proteins and then cleaved to give the active form, e.g. pro-caspases
    • Splice variants - alternative splicing may create different sized proteins produced from the same gene
    • Relative charge - the composition of amino acids (charged vs non-charged)
    • Multimers - e.g. dimerisation of a protein. This is usually prevented in reducing conditions, although strong interactions can result in the appearance of higher bands

    What is a clone number?

    A clone number is given to an antibody produced by a single clone of hybridoma cells. Each hybridoma cell clone produces one single pure homogeneous type of antibody. The term "monoclonal" pertains to a single clone of cells, a single cell and the progeny of that cell. Monoclonal antibodies can be made in large amounts in the laboratory.

    Each clone number represents a specific cell line cloned from ascites that was used to manufacture the antibody. Since antibodies are produced by more than one host, each cloned cell line receives a unique clone number. The clone number is not synonymous with the lot number which is in relation with the creation of the vial. There is little to no difference amongst the quality of the clones.

    Clone also refers to an individual developed from a single somatic (non-germ) cell from a parent, representing an exact replica of that parent. A clone is a group of cells derived from a single ancestral cell.

    What is a certificate of compliance (CoC)?

    Some of our antibodies are produced in-house and others are sourced from suppliers or academic collaborators whose products we believe will be of interest to our customers. All these products are guaranteed by our Abpromise to be of high quality. Our Abpromise assures you that if our products do not perform as our datasheets say they should we offer a replacement or refund within 12 months of purchase. Some of our collaborators are not certified ISO9001 and so we are not able to provide a certificate of analysis for these particular products.

    As the identity of our collaborators is confidential, we provide a certificate of compliance for all of our products available in our catalogue. This document certifies that our products meet the required specifications for a particular lot/batch of product.

    The Customer Services and Scientific Support teams would be very pleased to send a CoC on demand.

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