最近這幾次壓片出來的結果 都是空白片 覺得頭很大 不知道問題出在哪 有看版上先前討論過 二抗濃度的問題 自己並不是很清楚這之間的關係 @@ 第一次壓時有壓出明顯band來 之後想要modify 膠的濃度後 就再也沒壓出來過了 以下是我的方法過程 I run 12% SDS gel * 2 transfer 2hr 160mA block in 5% milk in PBST 4C overnight wash by PBST 5min * 3 1'Ab: anti-GST poly 1:2000 RT for 1.5hr wash by PBST 5min * 3 2'Ab: anti Rabbit 1:2500 RT for 1.5hr wash by PBST 5min * 3 detected by ECL kit the signal for 1min was too strong so, I keep the result of that for 30 sec II run 7% SDS gel transfer 2hr 80mA bloc in 5% milk in TBST(0.1%) 4C overnight wash by TBST 5min * 3 1'Ab: anti-GST poly 1:2000 RT for 1.5hr wash by TBST 5min * 3 2'Ab: anti Rabbit 1:2500 RT for 1.5hr wash by TBST 5min * 3 and detected by the same kit but there was no signal !! 再洗掉with TBST and rehybrid 1'Ab for 4'C overnight wash by TBST 5min * 3 2'Ab: anti Rabbit 1:2500 RT for 1.5hr wash by PBST 5min * 3 and detected by the same kit there were no signal again in both 5min and overnight transfer 確定有把protien 成功轉到membrane 上 所以目前推論有可能問題 如版上所說的二抗濃度過高? (為什麼) 但又為什麼第一次做的出來 有學長姊建議提高至1:2000?! TBST VS PBST 我查了 光TBST就有好多種配方 要用哪種才合適 一抗 二抗 壞掉~~ ?! 想破頭不知道問題在哪 害怕操作過程有問題也請學長在旁邊看(最後用kit時) 所以想請教各位 有可能的問題和改善方法 謝謝~ --

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