摘要
建立一种用 ＨＰＬＣ测定细胞中蛋白激酶活性的方法。方法：μＲｏｎｄｏｐｋａ Ｃ１８柱；流动相：甲醇：磷酸盐缓冲液（２０：８０ｖ／ｖ，内含５ ｍｍｏｌ／Ｌ ＰｉＣＡ，ｐＨ ７．０）；检测波长为２５９ ｎｍ；柱温：室温；灵敏度：０．０１ ＡＵＦＳ；进样 ２０ μｌ。结果：酶蛋白用量 ０． ３ ｍｇ左右、反应时间为 １０ ｍｉｎ、Ｈｉｓｔｏｎｅ（ Ⅲ－Ｓ）用量 １ｍｇ／ｍｌ、 ＡＴＰ浓度 ５００ μｍｏｌ／Ｌ时，活性测定结果理想。ＡＴＰ回收率为９９．６３％，ＲＳＤ为１．１１％（ｎ＝６）。结论：该方法简便、准确，适合于蛋白激酶的活性测定。
Purpose: The aim was to determine protein kinase activity in cells. Mathods: The chromatographic conditions were selected as follows: RP-C18 column, mobile phase included PiCA 5 mmol/L, methanol and KH2PO4 (20 : 80, v/v, pH v. 0), flow rate, 1. 0 ml/min, detected at 259 nm. Results: Good results can be obtained with the following conditions: the quantity of protein kinase is 0. 3 mg, the reactive time is 10 minutes, the concentration of Histone (Ⅲ-S) and ATP are 1 mg/ml and 500 μmol/L respectively. The recovery of ATP is 99. 63% with RSD 1. 11% (n= 6). Conclusion: This method is simple, accurate and reliable.
出处
《中国生化药物杂志》
CAS
CSCD
1999年第5期233-235,共3页
Chinese Journal of Biochemical Pharmaceutics
关键词
高效液相色谱
蛋白激酶C
蛋白激酶A
High performance liquid chromatography (HPLC), Protein kinase C, Protein kinase A
