Bethyl Laboratories, Inc.

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    Antibody Validation

    For more than 45 years, the scientific research community has trusted Bethyl Laboratories and our rigorous antibody validation process. Our scientists manufacture and validate all our antibodies at our lab in Montgomery, Texas, and if a product doesn’t meet our standards, it doesn’t leave our facility.

    At Bethyl, reproducibility matters and we understand its role within life science. As a result, we have devised a unique validation process that tests with paired antibodies raised against distinct protein epitopes. Validation is a continuous process at Bethyl, and we routinely evaluate new lots side-by-side with old lots to ensure lot-to-lot consistency.

    To pass our antibody validation process, products must meet four distinct criteria:

    • Specific: The antibody must bind specifically to the target protein.
    • Selective: The antibody must favor the target protein.
    • Reproducible: The antibody’s performance must be repeatable.
    • Sensitivity: The antibody must have acceptable levels of sensitivity for the detection of an endogenous target protein.


    The Validation Process

    Our two-phase process ensures that each antibody recognizes distinct epitopes of a target protein.

    Phase 1: Antigen Design

    Our antibody validation process begins with antigen design:

    1. We select between two and four peptides from distinct regions of the target protein.
    2. We immunize the hosts.
    3. We then antigen affinity purify the antibodies.

    At this stage, valid antibodies must agree in their recognition of the target protein. Agreement between the antibodies is determined in Phase 2.


    Phase 2: Validation for Specificity and Selectivity

    In the second phase of our antibody validation process, we test for specificity and selectivity using the western blot (WB) method in conjunction with immunoprecipitation (IP).

    1. We immunoprecipitate the endogenous target protein with each of the antibodies raised against distinct epitopes.
    2. We then immunoblot each of the immunoprecipitates with each of the epitope-distinct antibodies.
    3. If the antibody being tested is valid, it will recognize the immunoprecipitated protein of an antibody raised against an epitope from a different region of the protein.

    By analyzing western blots of immunoprecipitates, in conjunction with a western blot of the whole-cell lysate, we can verify the mobility of the target protein. Using multiple dilutions and a broad spectrum of whole-cell lysates, our scientists can verify selective binding as well as the antibody’s specificity, reproducibility, and sensitivity.

    At the conclusion of this two-phase process, only antibodies exhibiting the following characteristics qualify to be released:

    • Specific recognition of the target protein
    • Selective recognition of the target protein
    • Acceptable sensitivity
    • Reproducibility


    IHC Antibody Validation

    To evaluate antibody selectivity for immunohistochemistry (IHC), we use multiple antibodies generated against distinct epitopes to demonstrate concordance. Valid antibodies will produce similar staining patterns across a range of tissues and cells.

    1. Using two or more antibodies against distinct epitopes, we stain a broad spectrum of formalin-fixed paraffin-embedded (FFPE) tissues and cells.
    2. Once we establish selectivity, we determine the optimal conditions and dilutions for reproducible immunostaining.
    3. If more than one antibody indicating utility in IHC is not available, we confirm that the observed staining pattern agrees with published studies and curated data.


    To qualify for utility in IHC, antibodies must exhibit

    • Expected staining patterns
    • Concordant staining
    • Sensitivity
    • Selectivity
    • Reproducibility


    To learn more about Bethyl Laboratories’ antibody validation process, read our Application Note published in BioTechniques.

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    Guarantee Statement

    Bethyl's purpose is to improve lives by supporting scientific discovery. This drives our complete dedication to the highest standards of product and service quality.

    Bethyl is unique in our end-to-end control of the entire antibody lifecycle. From our veterinary facilities to the development, production and validation labs, the Bethyl team focuses on delivering quality products. This attention to quality at every step and the integration across each stage of the process means scientists can rely on Bethyl antibodies. We also offer expert technical support to help ensure your success.

    Recognizing that your research and time are valuable, we promise to stand behind our products. Should one of our antibodies fail to return satisfactory results when used in the species, application and under the same conditions stated on the product datasheet, Bethyl will offer a full product replacement, credit or refund at the investigator's discretion. Before providing product replacement, credit or refund, we ask the investigator to complete an application specific questionnaire and work with our Technical Support team to identify potential steps to optimize conditions.

    Every Bethyl product ships with a 100% guarantee; choose Bethyl antibodies with confidence.

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Bethyl Laboratories, Inc. has been dedicated to supporting scientific discovery through its qualified antibody products and custom polyclonal antibody services since its founding in 1972. Currently, Bethyl’s portfolio consists of over 9,500 catalog products; offering over 8,200 primary antibodies targeting over 3,300 proteins and 1,300 secondary antibodies raised against immunoglobulins from over 25 species. With over 45 years of experience, Bethyl is also a leading provider of custom polyclonal antibody production services. Bethyl offers complete packages from initial peptide synthesis to affinity purification of custom polyclonal antibodies from an antigen-specific immunosorbent. Most recently, our capabilities have expanded and we now offer contract recombinant rabbit monoclonal antibody partnerships. Every antibody that Bethyl sells has been manufactured to exacting standards at its sole location in Montgomery, Texas, and has been validated in-house by Bethyl’s team of scientists. From the veterinary facilities to the development, production and validation labs, the entire Bethyl team focuses on delivering quality products. What does it take for an antibody to pass Bethyl’s validation and quality control? In other words, what makes a really good antibody? First and foremost, an antibody must be shown to recognize the intended protein target. An antibody must also show high sensitivity with minimal cross-reactivity. To achieve these goals, Bethyl has devised a unique process for validating the specificity of its antibody products, which is critical to reproducibility of antibody-based experiments in life science. While many companies settle on using data from a single antibody, Bethyl tests with paired antibodies raised against distinct protein epitopes. Only after an antibody has been validated for specificity in immunoprecipitation and/or Western blot is it then tested in additional applications such as immunohistochemistry, immunocytochemistry, ChIP, and proximity ligation assay. Because of its rigorous validation process and high standards, Bethyl does not sell every antibody it makes. Bethyl serves to advance science by concentrating its resources on developing qualified antibodies, including many to emerging and underserved protein targets.