论文部分内容阅读
目的探索建立采用毛细管区带电泳(CZE)法分析血浆相关蛋白质的条件和方法。方法 1)以血浆COHN法组分Ⅳ(CFⅣ)抽提液为样品,代表蛋白质混合物,分别从波长、基本缓冲液、添加剂、PH、温度等方面,摸索CEZ分析蛋白质混合物的较优条件和方法;2)将摸索得到的较优条件下的CZE分析CFⅣ抽提液的结果,并与通过高效液相色谱(HPLC)法、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)法及双向电泳(2-DE)法分析CFⅣ抽提液的结果比较;3)采用较优条件下的CZE法分析标准血浆、标准血浆经离子交换层析(IEC)的FⅧ粗提液等,考察该条件是否适用于血浆中的其他组分。结果 1)CZE法分析CFⅣ抽提液的较优条件为:20℃以214 nm紫外吸收波长,0.5 psi压力进样10 s,10 kV恒压分析50 min,缓冲体系为pH 9.0的0.04 mol/L硼酸硼砂、0.05%SDS、0.007 5%腐胺;2)采用此条件的CZE法分析CFⅣ得到14个蛋白峰、分析时间50 min,HPLC法分析得到10个蛋白峰、分析时间60 min,SDS-PAGE法分析得到11条蛋白条带、分析时间120 min,2-DE法分析得到14个蛋白点数(与CZE分析的蛋白峰数数量相同)、分析时间3 d;3)CZE法用于标准血浆、标准血浆经离子交换层析的FⅧ粗提液的分析可分别分辩出22和11个蛋白峰。结论以CZE法分析血浆相关蛋白混合物与现有分析方法比较,能达到较好的分析效果,经摸索确定的较优分析条件具有一定的通用性。
Objective To establish a method and method for the analysis of plasma-related proteins by capillary zone electrophoresis (CZE). Method 1) The plasma COHN method extract of component Ⅳ (CF Ⅳ) samples for the representative of the protein mixture, respectively, from the wavelength, the basic buffer, additives, PH, temperature, and other aspects of CEZ analysis of protein mixtures of the better conditions and methods ; 2) The results of CFZ extracted by CZE under the optimal conditions were compared with the results of high performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE ) And two-dimensional electrophoresis (2-DE) analysis of CFⅣ extract comparison results; 3) CZE analysis of the standard plasma under the best conditions, the standard plasma by ion exchange chromatography (IEC) , To investigate whether this condition applies to other components in plasma. Results 1) The optimal conditions of CFⅣ extraction by CZE were as follows: UV absorption wavelength of 214 nm at 20 ℃, 10 s at 0.5 psi, constant voltage of 10 kV for 50 min, buffer system of 0.04 mol / L borax borate, 0.05% SDS and 0.007 5% putrescine; 2) CFⅣwas analyzed by CZE method to obtain 14 protein peaks. The analysis time was 50 min and 10 protein peaks were obtained by HPLC. The analysis time was 60 min. SDS -PAGE analysis of 11 protein bands, the analysis time of 120 min, 2-DE analysis of 14 protein spots (the same number of peaks with CZE analysis of protein), analysis time 3 d; 3) CZE method for the standard Analysis of plasma and standard plasma by FⅧ crude extract by ion exchange chromatography identified 22 and 11 protein peaks, respectively. Conclusion CZE analysis of plasma-related protein mixtures compared with the existing analysis methods, can achieve better analysis results, determined by the analysis of better analytical conditions has some versatility.