Marcon E, Jain H, Bhattacharya A, Guo H, Phanse S, Pu S, Byram G, Collins BC, Dowdell E, Fenner M, Guo X, Hutchinson A, Kennedy JJ, Krastins B, Larsen B, Lin ZY, Lopez MF, Loppnau P, Miersch S, Nguyen T, Olsen JB, Paduch M, Ravichandran M, Seitova A, Vadali G, Vogelsang MS, Whiteaker JR, Zhong G, Zhong N, Zhao L, Aebersold R, Arrowsmith CH, Emili A, Frappier L, Gingras AC, Gstaiger M, Paulovich AG, Koide S, Kossiakoff AA, Sidhu SS, Wodak SJ, Gräslund S, Greenblatt JF, Edwards AM
Nat. Methods 2015 Aug;12(8):725-31
PMID: 26121405
Abstract
Antibodies are used in multiple cell biology applications, but there are no standardized methods to assess antibody quality-an absence that risks data integrity and reproducibility. We describe a mass spectrometry-based standard operating procedure for scoring immunoprecipitation antibody quality. We quantified the abundance of all the proteins in immunoprecipitates of 1,124 new recombinant antibodies for 152 chromatin-related human proteins by comparing normalized spectral abundance factors from the target antigen with those of all other proteins. We validated the performance of the standard operating procedure in blinded studies in five independent laboratories. Antibodies for which the target antigen or a member of its known protein complex was the most abundant protein were classified as ‘IP gold standard’. This method generates quantitative outputs that can be stored and archived in public databases, and it represents a step toward a platform for community benchmarking of antibody quality.