Koide Laboratory - Antibody Validation

Background
Antibodies to histone posttranslational modifications (PTMs) are important tools in epigenetics research, particularly in the widely used chromatin immunoprecipitation (ChIP) experiments. We have developed a sensitive method for quantitatively characterizing polyclonal and monoclonal antibodies for histone PTMs in a ChIP-like format. Our method directly evaluates how efficiently an antibody captures a peptide corresponding a histone tail harboring a PTM, and it determines the dissociation constant (Kd) for the interactions of an antibody with peptides harboring cognate or off-target PTMs.

References: Nishikori S, Hattori T, Fuchs SM, Yasui N, Wojcik J, Koide A, Strahl BD Koide S.(2012) Broad ranges of affinity and specificity of anti-histone antibodies revealed by a quantitative peptide immunoprecipitation assay. J Mol Biol. 424:391. (PubMed); Hattori T, Taft JM, Swist KM, Luo H, Witt H, Slattery M, Koide A, Ruthenburg AJ, Krajewski K, Strahl BD, White KP, Farnham PJ, Zhao Y, Koide S. Recombinant antibodies to histone post-translational modifications. Nat Methods. 2013 Aug 18. (PubMed)

 

Tested Antibodies

Histone Mark Antibody Cat # Lot # Vendor Pass/Fail Specificity Affinity Binding Capacity Data
H3K4me1 07-436 DAM1687548 Millipore Fail Crossreact w/ K4me0 & K9me1 Low High J Mol Biol 424:391
H3K4me3 AM39159 1609004 ActiveMotif Pass Excellent High Low J Mol Biol 424:391
H3K9me3 Ab8898 GR36722 Abcam Fail Low sequence specificity Low High J Mol Biol 424:391
H3K9me3 Ab8899 960144 Abcam Fail Low sequence specificity Low High J Mol Biol 424:391
H3K9me3 Ab8899 339901 Abcam Fail Low sequence specificity Low High Nat Methods 2013
H3K9me3 05-1242 NG1800806 Millipore Fail No binding detected Extremely low Extremely low J Mol Biol 424:391
H3K9me3 9754 1 CST Fail Low sequence specificity High High Nat Methods 2013
H3K9me3 309-34839 11011 Wako Fail Low methylation specificity Low Low Nat Methods 2013
H3K9me3 pAb-056-050 A93-0042 Diagenode Pass Excellent High Low Nat Methods 2013
H3K9me3 pAb-056-050 A1675-001P Diagenode Fail Low sequence specificity High High Nat Methods 2013