Quantification of Radiolabeled Proteins in Polyacrylamide Gels
Autoradiography is often used to detect and quantify radiolabeled proteins present after separation by polyacrylamide gel electrophoresis (PAGE) (see Chapter 38). The method, however, requires relatively high levels of radioactivity when weak β-emitters, such as tritium, are to be detected. In addition, lengthy exposures requiring the use of fluorescent enhancers are often required. Recent developments in detection of proteins using silver staining (1,2) have added to the problem because of the fact that tritium emissions are quenched by the silver (2). Since for many metabolic labeling studies, tritium labeled precursors are often the only ones available, it seemed useful to develop a method that would overcome these drawbacks. The method the author developed involves the use of a cleavable crosslinking agent in the polyacrylamide gels that allows the solubilization of the protein for quantification by scintillation counting. Although developed for tritium (3), the method works well with any covalently bound label, as demonstrated here with 35S. Resolution is as good as or better than autoradiography (3 and Fig. 1), turnaround time can be greatly reduced, and quantification is more easily accomplished.
KeywordsSodium Dodecyl Sulfate Silver Nitrate Ammonium Persulfate Proper Polymerization Silver Stain