Verification of antibody specificity by peptide array is often performed for antibodies that are used to study epigenetics. Many of these antibodies are detecting specific post-translational modifications (PTMs) that regulate specific epigenetic states. Peptide array testing is one strategy we use to validate Invitrogen antibodies for research use.
The most common antibodies validated by peptide array are histones with specific modifications, such as phosphorylation, acetylation and methylation at specific amino acids. In addition, peptide arrays can be used to analyze the specificity of modification specific antibodies, such as the specificity of our acetyl lysine monoclonal antibody clone 1C6 (Cat. No. MA1-2021) for acetylated lysine on peptides.
Peptide array validation data
Histone peptides containing 384 different modification combinations spotted in duplicate on an array are used to verify antibody specificity. In a typical experiment, the array is probed with a modification specific antibody followed by a HRP-labeled secondary antibody and chemiluminescent detection. In the example below, an ABfinity rabbit oligoclonal (mono-) methyl-histone H3 (Lys4) antibody (Cat. No. 710795) was used to specifically detect histone H3 when methylated on lysine 4. The data show minor cross-reactivity to dimethylated lysine H3 and methylated histone lysine 20. The antibody did not detect other modifications of histone H3 or other histones and their modifications.
Peptide arrays performed using an ABfinity recombinant rabbit oligoclonal antibody (Cat. No. 710795) and two other suppliers’ antibodies targeting H3K4me1. Arrays were incubated overnight with a 1:2,000 dilution of primary antibody. After washing, the arrays were incubated with goat anti–rabbit IgG (H+L) Superclonal secondary antibody at a dilution of 1:5,000 for 1 hr. Arrays were incubated with SuperSignal West Pico substrate (Cat. No. 34078) and then visualized using a myECL Imager.
Verifying target specificity of Invitrogen antibodies using peptide arrays
Invitrogen antibodies that have been verified with peptide arrays are indicated with a “verified specificity” symbol in search results and on relevant product pages. The data showing the verification will be provided on each product page.
*The use or any variation of the word “validation” refers only to research use antibodies that were subject to functional testing to confirm that the antibody can be used with the research techniques indicated. It does not ensure that the product(s) was validated for clinical or diagnostic uses.