Protocol

Protein Microarrays

Volume 785 of the series Methods in Molecular Biology pp 45-54

Date:

Utilization of RNAi to Validate Antibodies for Reverse Phase Protein Arrays

  • Heiko MannspergerAffiliated withDivision of Molecular Genome Analysis, German Cancer Research Center (DKFZ)
  • , Stefan UhlmannAffiliated withDivision of Molecular Genome Analysis, German Cancer Research Center (DKFZ)
  • , Ulrike KorfAffiliated withDivision of Molecular Genome Analysis, German Cancer Research Center (DKFZ)
  • , Özgür SahinAffiliated withDivision of Molecular Genome Analysis, German Cancer Research Center (DKFZ) Email author 

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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.

Key words

Reverse phase protein arrays Antibody validation Antibody specificity Epidermal growth factor receptor RNAi siRNAs