[求救] western壓片全黑
各位版友們好
上個月做實驗都還順手，自從這個月出去玩了幾天回來
甚麼都變了...最糟糕的就是western鏡然壓不出東西
想請教各位，請幫我指點迷津
我的蛋白是從細胞收下的用RIPA buffer 定量30ug
1. 跑電泳90v 30min, 100v-110v跑到結束，
2. transfer 100v 90min
3. 10% milk blocking 1H
4. 1抗 1:500 O/N
5. TBST wash 3/10min
6. 2抗1:7000 1H
7. TBST wash 4/10min
8. ECL(P開頭那家的) 1min
9. 壓片3min 洗出來黑嘛嘛的
我要的位置再160kDa左右，但是什麼也沒有
之前一模一樣的條件有做出來阿
positive control壓的出來阿
有問題的壓片網址 http://ppt.cc/nb9n
想請教各位我的問題出在哪阿??謝謝
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