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 |