Activity staining of mammalian ribonuclease inhibitors after electrophoresis in sealed vertical slab polyacrylamide gels.
A method for detecting the activity of ribonuclease inhibitors (RIs) after nondenaturing polyacrylamide gel electrophoresis and isoelectric focusing was developed. Both types of electrophoresis were performed using vertical slab polyacrylamide gels in the presence of dithiothreitol and in a sealed system. In each system, purified 50 kDa human RI was visualized as a single band by immunoblotting with a specific antibody. RI activity in the polyacrylamide gel slab was detected by sandwiching the gel slab between a cellulose acetate membrane moistened with a solution of bovine pancreatic ribonuclease A and a dried agarose film sheet containing substrate yeast RNA plus ethidium bromide and incubating 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 formation. 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 band which compared favorably with that obtained on immunoblotting. These electrophoretic techniques appear useful for the investigation of RIs in heterogeneous biological samples.
Nadano
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D
Department of Legal Medicine, Fukui Medical School, Japan.
Yasuda
T
T
Takeshita
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H
Kishi
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K
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry; D005453:Fluorescence; D006801:Humans; D007525:Isoelectric Focusing; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000378:metabolism*; D010926:Placental Hormones / Q000276:immunology / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D011817:Rabbits; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D010926:Placental Hormones; C088463:ribonuclease inhibitor, brain; C022052:placental ribonuclease inhibitor; D012313:RNA; D012259:Ribonuclease, Pancreatic; D004996:Ethidium