小中大
同意二楼的建议,
不过还有一种可能性就是你的蛋白已经表达出来了,可能浓度太低,跑电泳不一定能跑出来,这样你就可以借助一些提高电泳灵敏度的技巧来试试了。
如:1、进染色方法,银染,最近看到一篇关于考染的改进方法,不错
传统的胶体考染法(Electrophoresis 1988,9,255-262)
染色液的成份(0.1% G-250, 10%(NH4)2SO4,2% H3PO4,20%甲醇)
[1] 固定:12%(W/V)三氯醋酸(TCA)2h
[2] 染色:200ML染色液混合50ML甲醇 16-24h(染色液:在490ML含2%的H3PO4中加入50g(NH4)2SO4直至完全溶解,再加0.5gCBBG250搅拌混合,定容至500ML使用前摇匀)
[3] 漂洗0.1mol/L Tris-H3PO4(PH6.5)漂洗两分钟;25%甲醇漂洗不超过1分钟
[4] 稳定:在20%的(NH4)2SO4中稳定蛋白质-染料复合物。
优点:背景低,灵敏度高,可达30ng protein/spot
改进的胶体考染法—Blue silver法(Electrophoresis 2004,25,1327-1333)
染色液的成份(0.12% G-250, 10%(NH4)2SO4,10% H3PO4,20%甲醇)
100ml H2O+100ml H3PO4+100g 粉末状(NH4)2SO4 先溶解,然后加入 1.2g G-250 再溶解,用水定容到800ml ,搅拌后加入200ml甲醇。
灵敏度可达1ng protein/spot
染色方法同上。
2、蛋白先浓缩再跑电泳,可以采用TCA沉淀法
If your protein is too dilute, you need to concentrate it before load on gel. Here is a method to concentrate protein before gel:
CHCl3 Precipitation of Proteins
For concentration of proteins from a variety of aqueous solutions.
In a 1.5-ml eppendorf tube take
0.1 ml of sample (or as much as possible)
add
0.4 ml of MeOH
0.1 ml of CHCl3
0.3 ml of H2O (optional)
This should give two phases upon centrifugation at 12,000g for 1 min. Protein should be precipitated at the interface. Pull off the upper phase leaving precipitated protein behind.
Add 0.3 ml of MeOH to give one phase.
Centrifuge at 12,000g for 2 min. Precipitated protein should pellet to bottom of tube.
Note: it is possible to scale up by 1.5x and still fit in a microfuge tube.
祝你成功!