Re: [問題] Western Blot

看板Biotech (生命科學)作者Mouseking (叫我老鼠王....-_-)時間19年前 (2006/11/22 20:38)推噓0(0推 0噓 0→)

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: 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？ -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 210.58.54.177