Sensitive autoradiographic quantification of electrophoretically separated proteases

A highly sensitive radioactive method has been developed to detect and quantify various molecular weight species of proteases in small volumes of crude biological samples. Following electrophoresis in a miniscale sodium dodecyl sulfate-polyacrylamide gel containing an iodinated protein substrate, the detergent is removed and the gel incubated to allow proteolysis. Radioactive degradation products formed in the gel are transferred onto nitrocellulose filter, and subsequently detected by autoradiography and/or counting. The transfer of radioactivity appears linear with respect to dose of protease. The method can be employed to quantitatively determine the size distribution of proteolytic activities in a crude biological sample, and to calibrate it according to a known standard. Activities as low as 0.5 munit of urokinase can be quantitatively measured, and activities 100-fold lower can be qualitatively detected by prolonged intensified autoradiography.