(192h) Refinement of an Internal Standard for Phosphotyrosine Western Blotting

Western blotting using either 1D or 2D sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D or 2D SDS PAGE) in combination with enhanced chemiluminescence detection (ECL) is the most sensitive method known for detecting specific proteins in complex mixtures. Our lab has been using an anti-phospho-tyrosine antibody for 1D and 2D SDS PAGE western blotting to visualize activated tyrosine kinases that might be functioning as cancer drivers in human tumor samples, pTyr-EGFR for example. One difficult problem is quantifying results. ECL light emission varies with time and with western blot conditions. Adding a pTyr-protein standard to every sample should allow normalization of results by determining the density ratio of unknown 1D bands or 2D spots to that of the internal standard. Last year we showed that Alk48 and Alk70 recombinant fragments could be phosphotyrosinylated in vitro with good results, but both pTyr-Alk proteins showed aggregation patterns during 2D gel electrophoresis. Their position was also affected by some samples. In this report, we show that blocking sulfhydral groups by alkylation with iodoacetyamide prevents aggregation patterns of the Alk48 fragment and stabilizes its position. Results are presented for use of the pTyr-Alk48-Iodoacetylated protein as an internal standard.