用western blotting方法检测乙醇固定细胞中细胞周期素的表达  

WESTERN BLOTTING ANALYSIS OF CYCLINS IN ETNANOL-FIXED CELLS

作　　者：冷彦[1] 陶德定[1] 申漫里[1] 周晖[1] 余源[1] 龚建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院分子医学中心,武汉430030

出　　处：《中国组织化学与细胞化学杂志》2001年第3期323-325,共3页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金 (No.396 70 36 5 ;39730 2 70 ;3972 5 0 2 7);卫生部科研基金 (No.2 0 2 - 0 1- 0 6 )资助课题

摘　　要：用 western blotting法测定乙醇固定细胞中细胞周期素 (cyclin)的表达情况 ,探索 western blotting和流式细胞仪对固定细胞在流式细胞术分选前或分选后对同批标本进行蛋白同步分析的可行性。采用对数生长期的 Molt- 4细胞 ,经两种常规固定方法固定后 ,用 western blotting方法检测 cyclin A、B1 、D和 E的表达。另取乙醇固定经流式细胞仪分选的 G1 期和 G2 / M期细胞裂解后 ,用 western blotting方法检测不同时相细胞中 cyclin B1 的表达。结果显示从固定细胞中提取的蛋白经 western blotting检测可得到清晰且分子量正确的条带 ,两种不同方法固定的细胞中检测到的 cyclins表达未见明显差异。乙醇固定细胞经分选后提取蛋白行 western blotting检测可见 G2 / M期细胞的 cyclin B1 有明显表达而 G1 期细胞不明显。细胞进行固定、洗涤、染色和分选等处理后不影响 western blotting对其 cyclins表达的分析 ,说明用 westernIn order to explore the possibility of cyclin analysis by flow cytometry combined with western blotting in cell cycle research.This study investigated with western blotting the expression of cyclins in ethanol fixed cells and their sorted cells. The expression of cyclin A, B 1, D and E in Molt 4 cells fixed with two different routine methods was analyzed with Western blotting. In addition, western blotting was also conducted to examine the expression of cyclin B 1 in cells sorted in G 1 phase and G 2/M phase after ethanol fixation. Clear and correct bands were observed with western blot analysis in the lysates from the fixed cells. There were no obvious differences in the levels of cyclins between the two fixation methods. Western analysis in the lysates collected from the sorted cells showed that the expression of cyclin B 1 was obvious in cells in G 2/M phase but not in cells in G 1 phase. The process of fixing, washing, dying and sorting the cells does not affect the assay of cyclins with western blotting. The results suggest that cyclin analysis using flow cytometry combined with western blotting is feasible.

关 键 词：western-blotting方法 检测 乙醇固定细胞 细胞周期素 表达 

分 类 号：Q26[生物学—细胞生物学] Q253