RNAi-based validation of antibodies for reverse phase protein arrays

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.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.The potential use of the RPPA technology in the field of proteomics and systems biology was introduced in 2001 by Paweletz and colleagues [1]. Since then, the RPPA technology has been further advanced, and successfully applied in numerous proteomic studies [2-13]. The basic principle of RPPA follows the idea of a dot-immunoblot; large numbers of samples are arrayed on solid phase carriers, and each array can then be probed with a different highly specific antibody. RPPA provide a semi-quantitative readout, and the expression of a particular target protein can be compared among all samples printed on a particular array. Printing of numerous replicate slides permits access to a highly parallelized analysis since each slide can be probed with a different detection antibody.The ou