最近这几次压片出来的结果 都是空白片 觉得头很大 不知道问题出在哪 有看版上先前讨论过 二抗浓度的问题 自己并不是很清楚这之间的关系 @@ 第一次压时有压出明显band来 之後想要modify 胶的浓度後 就再也没压出来过了 以下是我的方法过程 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 确定有把protien 成功转到membrane 上 所以目前推论有可能问题 如版上所说的二抗浓度过高? (为什麽) 但又为什麽第一次做的出来 有学长姊建议提高至1:2000?! TBST VS PBST 我查了 光TBST就有好多种配方 要用哪种才合适 一抗 二抗 坏掉~~ ?! 想破头不知道问题在哪 害怕操作过程有问题也请学长在旁边看(最後用kit时) 所以想请教各位 有可能的问题和改善方法 谢谢~ --

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