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Antibody Development and Validation Overview

Our antibody development process helps ensure that our products perform as specified. Antibody development and testing is a very important and tightly controlled process, with many steps along the way. From the design of the immunogen to the specificity screening and technique validation, our years of expertise in antibody development ensures that only the highest quality antibodies are offered for use in your research.

To see all of our primary antibodies, please go to the Antibodies page. Or, you can choose to see antibodies in various categories by selecting from the links in the top right.

Immunogen Selection

Immunogens are selected to decrease the likelihood of cross-reactivity with related proteins and to maximize detection of the protein in its native context. Immunogens for modification-specific antibodies are selected to ensure that the antibody recognizes the modified version of the protein only and does not cross-react with the same modification on different proteins.

PARP-1 protein domains.

Specificity screening

The first test performed on every antibody is dot blot analysis, which ensures its specificity for the desired protein or modification. Antibodies that do not exhibit a greater than 25-fold selectivity for the desired modification are failed.

Dot Blot.

Peptide array validation

For histone modification antibodies that require more stringent specificty testing, we utilize our MODified™ Histone Peptide Array to identify and quantitate any cross-reactivity to other modifications. The MODified Histone Peptide Arrays screen 59 modifications on the N-terminal tails of histones H2A, H2B, h2 and H4. Active Motif is the only company that performs this additional level of testing.

For a list of all antibodies tested using the MODified Histone Peptide Array, please click here.

Dot Blot.Histone h2 dimethyl Lys4 antibody specificity tested by peptide array analysis.

Western Blot

Western blotting is performed to verify the antibody recognizes a protein of the correct molecular weight and does not cross-react with other proteins. Commonly used chemical treatments that stimulate a specific modification (e.g. HDAC inhibitors and acetylation) are used to better detect the modification.

Western Blot.

Technique Validation

Each antibody is validated for use in important techniques such as chromatin immunoprecipitation (ChIP) and immunofluorescence (IF), so that you have confidence when using them in your experiments.


Additional Specificity Testing

Antibodies to histone modifications that exist in budding yeast (S. cerevisiae) can be further screened for specificity by testing strains of yeast that contain a point mutation at the position of the modified amino acid.

Western Blot 2