[問題] Western Blot

看板Biotech (生命科學)作者janmei (展眉)時間19年前 (2006/11/22 20:05)推噓0(0推 0噓 0→)

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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~ -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.109.40.10