用western blotting方法检测乙醇固定细胞中细胞周期素的表达

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cyclinA,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cyclinB1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cyclinB1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进行蛋白同步分析是可行的。

WESTERN BLOTTING ANALYSIS OF CYCLINS IN ETNANOL-FIXED CELLS

In 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.