RNAi-based validation of antibodies for reverse phase protein arrays.

ABSTRACT: BACKGROUND: Reverse phase protein arrays (RPPA) have been demonstrated to be a useful experimental platform for quantitative protein profiling in a high-throughput format. Target protein detection relies on the readout obtained from a single detection antibody. For this reason, antibody specificity is a key factor for RPPA. RNAi allows the specific knockdown of a target protein in complex samples and was therefore examined for its utility to assess antibody performance for RPPA applications. RESULTS: To proof the feasibility of our strategy, two different anti-EGFR antibodies were compared by RPPA. Both detected the knockdown of EGFR but at a different rate. Western blot data were used to identify the most reliable antibody. The RNAi approach was also used to characterize commercial anti-STAT3 antibodies. Out of ten tested anti-STAT3 antibodies, four antibodies detected the STAT3-knockdown at 80-85%, and the most sensitive anti-STAT3 antibody was identified by comparing detection limits. Thus, the use of RNAi for RPPA antibody validation was demonstrated to be a stringent approach to identify highly specific and highly sensitive antibodies. Furthermore, the RNAi/RPPA strategy is also useful for the validation of isoform-specific antibodies as shown for the identification of AKT1/AKT2 and CCND1/CCND3-specific antibodies. CONCLUSIONS: RNAi is a valuable tool for the identification of very specific and highly sensitive antibodies, and is therefore especially useful for the validation of RPPA-suitable detection antibodies. On the other hand, when a set of well-characterized RPPA-antibodies is available, large-scale RNAi experiments analyzed by RPPA might deliver useful information for network reconstruction.

SUBMITTER: Mannsperger HA 

PROVIDER: S-EPMC3022873 | biostudies-other | 2010

REPOSITORIES: biostudies-other