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人APL细胞株NB4不同二维电泳条件对电泳结果的影响
引用本文:张群业,黄秋花,沈树红,王海红,陈赛娟,陈竺. 人APL细胞株NB4不同二维电泳条件对电泳结果的影响[J]. 中国实验血液学杂志, 2004, 12(4): 401-405
作者姓名:张群业  黄秋花  沈树红  王海红  陈赛娟  陈竺
作者单位:上海第二医科大学附属瑞金医院上海血液学研究所,医学基因组学国家重点实验室,上海,200025
基金项目:国家重点基础研究发展规划 ( 973 )资助项目( 2 0 0 1CB5 10 2 0 8)
摘    要:本研究探讨不同二维电泳条件对人急性早幼粒细胞性白血病(APL)细胞株NB4二维电泳结果的影响。选用24cm,pH3—10L的固定pH梯度(immobilized pH gradient,IPG)胶条,用IPGphor进行第一相等电聚焦,然后用Ettan Dalt Ⅱ垂直电泳系统进行第二相SDS-PAGE,银染后用Image Master 2D Elite软件进行分析。结果表明:采用低离子强度的缓冲液冲洗收集样品可以提高等电聚焦效果。裂解液中7mol/L尿素、40mmol/L DTT能溶解NB4细胞大部分蛋白质,而水化液中硫脲与尿素合用可以明显增加碱性端中低分子量区可分辨的蛋白质点。NB4细胞用24cm,pH3—10L的IPG胶条进行二维电泳时,100μg左右的上样量、63200V小时左右的等电聚焦时间是恰当的。结论:APL细胞的蛋白质种类繁多,受多种因素影响,正确选择样品制备方法和二维电泳条件十分重要。

关 键 词:急性早幼粒细胞白血病 蛋白质组 NB4细胞株 二维电泳 IPG
文章编号:1009-2137(2004)04-0401-05
修稿时间:2003-09-22

Influence of Different Conditions on Two-Dimensional Gel Electrophoresis of Proteins from Human Acute Promyelocytic Leukemia Cell Line NB4
ZHANG Qun-Ye,HUANG Qiu-Hua,SHEN Shu-Hong,WANG Hai-Hong,CHEN Sai-Juan,CHEN Zhu State Key Laboratory of Medical Genomics. Influence of Different Conditions on Two-Dimensional Gel Electrophoresis of Proteins from Human Acute Promyelocytic Leukemia Cell Line NB4[J]. Journal of experimental hematology, 2004, 12(4): 401-405
Authors:ZHANG Qun-Ye  HUANG Qiu-Hua  SHEN Shu-Hong  WANG Hai-Hong  CHEN Sai-Juan  CHEN Zhu State Key Laboratory of Medical Genomics
Affiliation:State Key Laboratory of Medical Genomics, Shanghai Institute of Hematology, Rui Jin Hospital, Shanghai Secondary Medical University, Shanghai 200025, China.
Abstract:The aim of this study was to explore the effect of different conditions on two-dimensional gel electrophoresis of proteins from human acute promyelocytic leukemia cell line NB4. The 24 cm pH 3-10 linear immobilized pH gradient (IPG) strips were chosen, the isoelectric focusing was carried out by using IPGphor. Then, the second-dimensional SDS-PAGE was performed. After silver staining, the gel was analyzed by ImageMaster 2D Elite. The results showed that low ion intensity sample washing buffer improved the performance of isoelectric focusing. The lysis buffer containing 7 mol/L urea and 40 mmol/L DTT could solubilize the most proteins from NB4 cell line. The rehydration solution containing thiourea and urea increased the low molecular weight protein points to be resolved in the area of basic end. The reasonable sample load and Volt/hour of NB4 were about 100 micro g and 63 200 V/h for the 24 cm pH 3-10 IPG strips. It is concluded that the proteins from NB4 and similar cell line are complicated and affected by many factors, so that, it is very important to select the right methods for sample preparation and the conditions of two-dimensional gel electrophoresis.
Keywords:APL  Proteomics  NB4 cell line  Two-dimensional gel electrophoresis  IPG
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