: 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 : 第一个问题....你的percentage为何可以差这麽多7% v.s 12%? 还有transfer 的电流？如果gel大小一样为何要减半？ : 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?! 二抗太高只会压海苔....你的没signal band不必考虑降低二抗！ : TBST VS PBST 这个问题有深度.....当初也困扰我很久...... 我问过很多做Western好的lab同学....其实他们也不知道 怎回答这问题...TBST tris-base saline buffer加 tween 20 而PBST是phosphate-base buffer saline加 triton x-100 我当年是搞不清楚tween 20 和Triton X-100有何不同？ 经过这几年....我的结论是....黑猫花猫白猫....会抓老鼠就是好猫 一个大原则是做磷酸化抗体别用PBST....其他都是实徵理由... 如果有错希望高手指导....所以建议你自己做个实验PBST TBST side by side跑一次.....证实看看是不是PBST才行？ --

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