(101a) Characterization of an Internal Standard for Phosphotyrosine Western Blotting

Characterization of an Internal Standard for Phosphotyrosine Western Blotting

Western blotting in combination with carrier ampholine 2D gel electrophoresis (CA-2D) and enhanced chemiluminescence detection (ECL) is the most sensitive method known for detecting specific proteins in complex mixtures. Our lab has been using an anti-phosphotyrosine antibody in combination with CA-2D western blotting to visualize specific receptor tyrosine kinases (RTK) in human tumor samples. Active RTKs, which are cancer drivers, have multiple phosphotyrosines (pTyr). The same RTK without pTyr is inactive, EGFR for example. One problem we have encountered however, is in quantifying results. The time course of ECL light emission varies dramatically over the first hour. Thus the same exposure time, 3 min for example, gives a different visual pattern at the beginning of the hour than at the end for the same sample. One way of quantifying the western blot reaction is to include an internal standard. Results from the spot density of the protein of interest are then normalized by expression as a ratio of the spot density of the standard. Surprisingly few pTyr standards are available. In this talk we will discuss results from three potential pTyr internal standards: JNK1 protein from the University of Dundee, phosphotyrosinylated BSA from Sigma, and phosphotyrosinylated ALK fragment from ProQuinase.