ACTIVITY STAINING OF MAMMALIAN RIBONUCLEASE INHIBITORS AFTER ELECTROPHORESIS IN SEALED VERTICAL SLAB POLYACRYLAMIDE GELS

A method for detecting the activity of ribonuclease inhibitors (RIs) a fter nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed u sing vertical slab polyacrylamide gels in the presence of dithiothreit ol and in a sealed system, In each system, purified 50 kDa human RI wa s visualized as a single band by immunoblotting with a specific antibo dy. RI activity in the polyacrylamide gel slab was detected by sandwic hing the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose fil m sheet containing substrate yeast RNA plus ethidium bromide and incub ating at 37 degrees C. The ribonuclease penetrated the polyacrylamide gel and digested the substrate RNA in the agarose film. However, if an RI was present in the gel, the enzyme was inactivated by complex form ation. Fluorescent bands corresponding to RIs were observed on a dark background under ultraviolet light. This activity staining had a high sensitivity allowing detection of less than 0.6 units of mammalian RIs (corresponding to 5 ng of purified human RI) and produced a sharp ban d which compared favorably with that obtained on immunoblotting, These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples. (C) 1995 Academic Press, Inc.