三氯醋酸/丙酮沉淀法与硫酸铵沉淀法去除血浆高丰度蛋白效果的对比研究  

Comparative study on the effect of TCA/acetone-precipitation and ammonium sulfate precipitation in removing plasma high-abundance protein

作　　者：吴天鸽 黄文君[1] 冯嘉轩 WU Tiange;HUANG Wenjun;FENG Jiaxuan(Department of Blood Transfusion,the Second Hospital of Jilin University,Changchun,Jilin 130000,China;Experimental Center of Clinical Medical College,Changchun University of Traditional Chinese Medicine,Changchun,Jilin 130000,China)

机构地区：[1]吉林大学第二医院输血科,长春130000 [2]长春中医药大学临床医学院实验中心,长春130000

出　　处：《重庆医学》2020年第23期3876-3879,共4页Chongqing medicine

摘　　要：目的探讨三氯醋酸(TCA)/丙酮沉淀法与硫酸铵沉淀法去除血浆高丰度蛋白的效果。方法选取2017年6月至2019年6月长春市中心血站采集的O型RHD阳性新鲜冰冻血浆12例,组成4个样品并分别编号为A1、A2、B1、B2。采用TCA/丙酮沉淀法去除A1、A2样品血浆中高丰度蛋白,硫酸铵沉淀法去除B1、B2样品血浆中高丰度蛋白,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)验证血浆中高丰度蛋白去除的效果。Clean-up Kit去除蛋白质中干扰物质,并采用SDS-PAGE和双向电泳(2-DE)验证Clean-up Kit的效果。结果银染的SDS-PAGE胶图显示,TCA/丙酮沉淀法与硫酸铵沉淀法均可有效去除血浆高丰度蛋白,但与硫酸铵沉淀法相比,TCA/丙酮沉淀法的SDS-PAGE条带更清晰。SDS-PAGE和2-DE显示两种方法去除清蛋白的血浆Clean-up Kit后对去除血浆中其他干扰物质效果并不明显。结论TCA/丙酮沉淀法去除血浆高丰度蛋白的效果更好,且去除血浆中清蛋白后的血浆Clean-up Kit对去除效果无显著影响,可直接进行2-DE、图像分析及质谱检测分析。Objective To explore the difference in the effect of trichloroacetic acid(TCA)/acetone precipitation and ammonium sulfate precipitation on the removal of plasma high-abundance protein.Methods The subjects were selected from 12 cases of O-type RHD-positive fresh frozen blood collected from the blood station from June 2017 to June 2019.4 samples were composed and numbered A1,A2,B1 and B2 respectively.The high-abundance protein in the plasma of A1 and A2 samples was removed by TCA/acetone precipitation.which of B1 and B2 samples was removed by ammonium sulfate precipitation.SDS-PAGE was performed to verify the effectiveness of high-abundance protein removal in plasma.The Clean-up Kit removes interfering substances from proteins and its effect validated using SDS-PAGE and two-dimensional electrophoresis(2-DE).Results Silver-stained SDS-PAGE gel shows that both TCA/acetone precipitation and ammonium sulfate precipitation can effectively remove plasma high-abundance protein,but compared with ammonium sulfate precipitation,the belts of TCA/acetone precipitation SDS-PAGE is clearer.After Clean up after removing plasma albumin with the two methods,SDS-PAGE and 2-DE suggested that the effect of removing other interfering substances in plasma is not obvious.Conclusion TCA/acetone precipitation method has a better effect on removing plasma high-abundance protein,and Clean up Kit after removing has no significant effect on the removal effect,and can directly perform 2-DE,image analysis and mass spectrometry detection.

关 键 词：三氯醋酸/丙酮沉淀法 饱和硫酸铵沉淀法 血浆 高丰度蛋白 

分 类 号：R446.11[医药卫生—诊断学]