pH gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: Detection and inhibition of enzyme activity in the gel

✍ Scribed by Daita Nadano; Toshihiro Yasuda; Kazumi Sawazaki; Haruo Takeshita; Koichiro Kishi

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 637 KB
• Volume: 17
• Category: Article
• ISSN: 0173-0835
• DOI: 10.1002/elps.1150170118

✦ Synopsis

pH Gradient electrophoresis of basic ribonucleases in sealed slab polyacrylamide gels: Detection and inhibition of enzyme activity in the gel A simple method for the separation and specific detection of basic ribonucleases (RNases) was developed. The separation was achieved by polyacrylamide gel electrophoresis in a pH gradient generated by a carrier ampholyte (Pharmalyte 8-10.5) and arginine. In order to prevent interference from atmospheric carbon dioxide, the pH gradient was formed in sealed vertical gel slab. Human nonsecretory-type RNase, bovine pancreatic RNase A, and other basic proteins could be resolved without expensive equipment or complicated procedures. For activity detection after electrophoresis a zymogram technique was applied, using dry agarose film containing ethidium bromide plus RNA as sub- strate. This approach affords two advantages: (i) Basic RNase activities can be detected within 15 min, even in crude materials. The sensitivity is better than 0.5 ng of purified human nonsecretory-type RNase. (ii) An inhibition test of RNase activities in the gel, using human placental-type RNase inhibitor, can be performed.