Nadano D, Yasuda T, Sawazaki K, Takeshita H, Kishi K
Department of Legal Medicine, Fukui Medical School, Japan.
Electrophoresis. 1996 Jan;17(1):104-9. doi: 10.1002/elps.1150170118.
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 substrate. 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.
开发了一种分离和特异性检测碱性核糖核酸酶（RNases）的简单方法。分离是通过在由载体两性电解质（Pharmalyte 8 - 10.5）和精氨酸产生的pH梯度中进行聚丙烯酰胺凝胶电泳来实现的。为了防止大气二氧化碳的干扰，pH梯度在密封的垂直凝胶板中形成。人非分泌型RNase、牛胰核糖核酸酶A和其他碱性蛋白质无需昂贵设备或复杂程序即可分离。电泳后进行活性检测时，采用含有溴化乙锭加RNA作为底物的干琼脂糖膜的酶谱技术。这种方法有两个优点：（i）即使在粗样品中，碱性RNase活性也能在15分钟内检测到。灵敏度优于0.5 ng纯化的人非分泌型RNase。（ii）可以使用人胎盘型RNase抑制剂对凝胶中的RNase活性进行抑制试验。
