正丁醇法制备大鼠脂肪组织细胞膜TSHR

目的:利用正丁醇对大鼠脂肪组织细胞膜上的促甲状腺素受体(TSHR)进行提纯,以获得膜受体的均一水溶性溶液。方法:使用差速离心法获得大鼠含TSHR脂肪组织细胞膜碎片后,用正丁醇进一步提纯;采用SDS-PAGE和ELISA法鉴定所得蛋白完整性、生物学活性,并用BCA法测定相关蛋白含量。结果:正丁醇提纯的大鼠TSHR结构完整;经正丁醇提纯后的TSHR包被酶标孔孔间测定值变异系数较未经正丁醇提取组以及阳性对照组低。结论:经正丁醇提纯后的大鼠TSHR可以满足临床ELISA法检测Graves病患者血清TRAb的要求,同时可以降低酶标孔间测定值的误差。

Extraction of thyrotropin receptors from membrane preparations of the fat tissue of male rats with n-butanol

Objective: To develop a new method for extracting thyrotropin receptors(TSHR)from membrane preparations of the epididymal fat pads of male rats to get well-distributed water-solution.Methods: The crude particulate fraction containing rat fat cell membranes were prepared with differential centrifugation,then extracted with n-butanol.The integrity and bioactivity of TSHR from rat fat cell membrane were analyzed by means of SDS-PAGE and ELISA.Protein concentration was determined by using bicinchoninic acid solution(BCA)assays and BSA used as standard.Results:As desired,after extracted with n-butanol,TSHR didn't lose it's integrity.Well's CV of extracted TSHR group was the lowest amang 3 groups(unextracted,extracted,positive control).Conclusion:It is a useful methed to extract TSHR from a crude particulate fraction of rat fat cell membranes.And the TSHR can be well used in ELISA to detect TRAb for Graves disease patients.