Bethyl antibodies are highly regarded for passing strict validation testing before arriving in customers’ hands. Bethyl believes customers can make a more confident purchase when they know how Bethyl antibodies are validated. Read more in this article about the six pillars Bethyl applies in the lab every day for antibody validation.

Independent antibodies

Pillar 1 of Bethyl Laboratories Antibody Validation

This pillar requires that two or more antibodies directed against different epitopes of a protein generate similar results.  For example, two antibodies targeting different epitopes on a given protein target should produce similar staining patterns on the same tissue in an immunohistochemistry (IHC) study or produce similar results on a western blot. The majority of Bethyl antibodies are validated using independent antibodies developed in-house.

Example of data for this pillar (click to enlarge):

Bethyl Independent Antibodies

Complementary Assays

Pillar 2 of Bethyl Laboratories Antibody Validation

This pillar requires that multiple, antibody-dependent assays produce complementary results. For example, when the same antibody of interest produces cell line-specific staining in an immunocytochemistry (ICC) assay and shows similar specificity on a western blot. Additional antibody-dependent assays under this pillar of Bethyl include immunoprecipitation (IP), immunohistochemistry (IHC), flow cytometry, chromatin immunoprecipitation (ChIP), and competitive ELISA. This pillar also captures the second half of a reciprocal IP experiment—for example, an IP with an independent antibody (Pillar 1), followed by western blotting with the antibody of interest.

Example of data for this pillar (click to enlarge):

Orthogonal Characteristics

Pillar 3 of Bethyl Laboratories Antibody Validation

This pillar requires that antibody-independent and antibody-dependent assays produce results that are correlative. The antibody-independent techniques typically involve mass spectroscopy or mRNA transcript analysis of cell lines and tissue samples. Bethyl Laboratories retrieves this data using both proprietary databases and public databases, such as GeneVisible or ProteomicsDB. Bethyl looks for a clear relationship between these orthogonal data and western blot or immunocytochemistry (ICC) data generated using our antibodies in specific cell lines.

Example of data for this pillar (click to enlarge):

Bethyl Orthogonal Characteristics

Biological Characteristics

Pillar 4 of Bethyl Laboratories Antibody Validation

This pillar takes advantage of the unique biology associated with some protein targets. To satisfy this pillar, the antibody of detects a protein target (or post-translational modification) that is only expressed or present under specific conditions (e.g., hypoxia, drug treatment). A phosphatase treatment on a phosphorylated protein can also be utilized to show phospho-specificity. Bethyl commonly uses this pillar to validate phospho-specific antibodies.

Example of data for this pillar (click to enlarge):

Bethyl Biological Characteristics

Protein OE/Epitope Tags

Pillar 5 of Bethyl Laboratories Antibody Validation

This pillar uses over-expressed (OE) proteins to validate antibodies against targets where Bethyl Laboratories cannot identify a natively expressing cell line, or the protein is expressed at levels insufficient for detection. Bethyl also uses this pillar to rule out cross-reactivity with specific proteins. This pillar is always combined with one or more pillars.

Example of data for this pillar (click to enlarge):

Bethyl Protein OE-Epitope Tags

Genetic Strategies

Pillar 6 of Bethyl Laboratories Antibody Validation

This pillar uses gene knockout or knockdown to reduce the levels of target protein available for detection. For example, a knockout of a target protein should ideally result in a reduced band intensity on a western blot, or an abrogation of signal in those cells in an immunocytochemistry (ICC) experiment. This is not a commonly used pillar for Bethyl antibodies.

Example of data for this pillar (click to enlarge):

Bethyl Genetic Strategies