Electrophoresis in polyacrylamide gels is widely used for analysis and characterization of proteins and mixtures of proteins. It is frequently necessary to obtain estimates of the radioactivity of protein bands in the gels, and of the relative amounts of two radionuclides, generally 3H ,and l*C. Cain and Pitney (1) and Gray and Steffensen (2) recently reviewed several solutions to this problem. We have developed a new method which gives reliable results with a minimum of complex equipment, and without the necessity for expensive chemicals. To achieve this, slices of gels are treated with aqueous NH,OH on glass-fiber discs. Much of the protein diffuses out of the gel. The entire disc, including the remains of the gel slice is then dried and counted in a toluene/ PPO/POPOP scintillant.
Materials. Radioactive cell proteins (specific activities of 0.03 $Zi of 14C and 0.07 ,JZi of 3H per mg protein) used to test the counting procedures were obtained from BHK-Cl3 cells (3). Cells grown in a medium containing either 14C-or 3H amino acids were disrupted, and proteins were extracted with phenol from the supernatant fraction after centrifugation for 10 min at 18,000g.
Radioactive viral proteins were obtained from vesicular stomatitis virus, purified from infected BHK cells by the method of Cartwright, Smale, and Brown (4). To solubilize the proteins the purified virus was treated with acetic acid, SDS, and urea according to Summers, Maize& and Darnell (5). Before coelectrophoresis with viral protein, the phenolextracted cell protein was treated in the same way.
Unless otherwise stated, the gels used in this work contained 7.5% acrylamide, 0.2% N,N'-methylenebisacrylamide, 0.125% tetramethylenediamine, 0.0770 sodium persulfate, 0.0038% potassium ferricyanide