: 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
:
第一個問題....你的percentage為何可以差這麼多7% v.s 12%?
還有transfer 的電流？如果gel大小一樣為何要減半？
: 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?!
二抗太高只會壓海苔....你的沒signal band不必考慮降低二抗！
: TBST VS PBST
這個問題有深度.....當初也困擾我很久......
我問過很多做Western好的lab同學....其實他們也不知道
怎回答這問題...TBST tris-base saline buffer加 tween 20
而PBST是phosphate-base buffer saline加 triton x-100
我當年是搞不清楚tween 20 和Triton X-100有何不同？
經過這幾年....我的結論是....黑貓花貓白貓....會抓老鼠就是好貓
一個大原則是做磷酸化抗體別用PBST....其他都是實徵理由...
如果有錯希望高手指導....所以建議你自己做個實驗PBST TBST
side by side跑一次.....證實看看是不是PBST才行？
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