D. Nadano et al., ACTIVITY STAINING OF MAMMALIAN RIBONUCLEASE INHIBITORS AFTER ELECTROPHORESIS IN SEALED VERTICAL SLAB POLYACRYLAMIDE GELS, Analytical biochemistry, 227(1), 1995, pp. 210-215
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.