Measuring the Radioactivity of 2-D Protein Extracts

The in vivo radioactive labeling of proteins is used to enhance the sensitivity of detection and to quantitate the abundance individual proteins. To compare the quantity and identity of resolved proteins using 2-D gels, identical amounts of sample must be loaded. It is therefore a necessity to measure accurately the radioactivity of 2-D protein extracts to determine the amount of each sample to load onto the 2-D gel. The protocol for measuring the number of counts is achieved by differentially precipitating the protein products with trichloroacetic acid (TCA) from the unincorporated radioactive precursor, washing away the precursor, and measuring the radioactivity of the precipitate using a scintillation counter. This chapter provides two protocols for quantifying the incorporation of radioisotopes during in vivo labeling. The first protocol filters the TCA-precipitated proteins onto a glass filter fiber and the second directly measures the TCA-precipitated proteins. This chapter is provided as a handy reference for the novice wishing to run 2-D gels using radiolabeled cell extracts.