title: Utilization of RNAi to validate antibodies for reverse phase protein arrays.
abstract: Reverse phase protein arrays (RPPAs) emerged as a very useful tool for high-throughput screening of protein expression in large numbers of small specimen. Similar to other protein chemistry methods, antibody specificity is also a major concern for RPPA. Currently, testing antibodies on Western blot for specificity and applying serial dilution curves to determine signal/concentration linearity of RPPA signals are most commonly employed to validate antibodies for RPPA applications. However, even the detection antibodies fulfilling both requirements do not always give the expected result. Chemically synthesized small interfering RNAs (siRNAs) are one of the most promising and time-efficient tools for loss-of-function studies by specifically targeting the gene of interest resulting in a reduction at the protein expression level, and are therefore used to dissect biological processes. Here, we report the utilization of siRNA-treated sample lysates for the quantification of a protein of interest as a useful and reliable tool to validate antibody specificity for RPPAs. As our results indicate, we recommend the use of antibodies which give the highest dynamic range between the control siRNA-treated samples and the target protein (here: EGFR) siRNA-treated ones on RPPAs, to be able to quantify even small differences of protein abundance with high confidence.
authors: Division of Molecular Genome Analysis, German Cancer Research Center (DKFZ), Heidelberg, Germany.
authors: Heiko
authors: H
authors: Mannsperger
authors: Stefan
authors: S
authors: Uhlmann
authors: Ulrike
authors: U
authors: Korf
authors: Ozgür
authors: O
authors: Sahin
mesh_terms: D000906:Antibodies; D000918:Antibody Specificity; D015153:Blotting, Western; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D066246:ErbB Receptors; D006801:Humans; D040081:Protein Array Analysis / Q000295:instrumentation / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000138:chemical synthesis
chemical_list: D000906:Antibodies; D034741:RNA, Small Interfering; D066246:ErbB Receptors