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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.

View Bethyl's Immuno-Oncology Targets

In the world of next generation immuno-oncology research, having confidence in your immunoassay results is vital. Unfortunately, 75% of antibodies in today’s market are non-specific or simply do not work at all. That’s why at Bethyl, we manufacture and validate every antibody on-site to ensure target specificity and sensitivity. More than 10,000 independent citations over the past 15 years have proven that our antibodies will function as designed in your assay — and we offer a 100% guarantee.