Dr.Chen Gao cg_npwr@yahoo

1. 聚丙烯酰胺圆盘电泳分离血清蛋白 Dr.Chen Gao cg_npwr@yahoo.com.cn CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

2. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

3. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

4. 聚丙稀酰胺凝胶电泳（PAGE） • 目前分离分子的最好方法 • 以物质的物理差别，分子大小和净电荷为基础 • 还有分子筛的特殊作用，为分开电泳率很相近的大分子提供简单而有效的方法 • 凝胶孔径大小可通过凝胶浓度调节，机械强度好，弹性大，不产生电渗，化学惰性 • 需要样品量少并不易扩散 • 使用设备简单等 缺点 • 聚合反应必须用高纯度的试剂在没有空气的情况下控制其影响因素来实现 • 丙稀酰胺毒性大 CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

5. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

6. TEMED催化AP生成硫酸自由基： S2O82－ 2SO4·¯ 硫酸自由基的氧原子激活Acr单体并形成单体长链： Bis将单体长链间连成网状结构： AP-TMTED催化体系 CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

7. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

8. 化学聚合的凝胶孔径较小，常用于制备分离胶，重复性好。聚合反应受各种因素的影响：化学聚合的凝胶孔径较小，常用于制备分离胶，重复性好。聚合反应受各种因素的影响： •催化剂和加速剂的浓度 应选择合适的AP和TEMED浓度使聚合时间控制在30—60min内较好，过量的催化剂和加速剂会引起烧胶和蛋白质条带的畸变。 •pH TEMED只能以游离碱的形式发挥作用，在酸性条件下，叔胺缺少自由碱基，引发AP产生自由基的过程会被延迟，聚合时间延长。 •温度 聚合速度与温度有关，一般是高温聚合快，而聚合的速度影响交联孔径的大小，所以凝胶聚合时必须保持温度恒定，通常用与电泳相同的温度。 •分子氧 分子氧的存在会阻止碳链的延长，妨碍聚合作用，在聚合过程中要尽量避免接触空气。 •杂质 某些金属离子或其它杂质也会影响凝胶的化学聚合，所以应选择高纯度的Acr、Bis和AP。 CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

9. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

10. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

11. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

12. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

13. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

14. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

15. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

16. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

17. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

18. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

19. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

20. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

21. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

22. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

23. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

24. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

25. 结果示意图： γ β α2 α1 清蛋白 CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka

26. CECS 694-02 Introduction to Bioinformatics University of Louisville Spring 2004 Dr. Eric Rouchka