Antibody Validation
For more than 40 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.
Validation is a continuous process at Bethyl Laboratories, 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:
- We select between two and four peptides from distinct regions of the target protein.
- We immunize the hosts.
- 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).
- We immunoprecipitate the endogenous target protein with each of the antibodies raised against distinct epitopes.
- We then immunoblot each of the immunoprecipitates with each of the epitope-distinct antibodies.
- 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.
- Using two or more antibodies against distinct epitopes, we stain a broad spectrum of formalin-fixed paraffin-embedded (FFPE) tissues and cells.
- Once we establish selectivity, we determine the optimal conditions and dilutions for reproducible immunostaining.
- 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
