作者renshiue (UW-Madison)
看板Biotech
標題Re: [求救] 誘導表現蛋白質
時間Fri May 11 07:31:32 2007
※ 引述《content71 (羅莉飼養中...)》之銘言：
: E. coli strain : BL21(DE3)
: BL21(DE3)pLysS
: Rosetta(DE3)
: induction : E. coli OD600 ~0.4
: 0.5mM, 1mM, for 37度C, 3hr
: result --> BL21(DE3) 在induction後就不太生長，SDS-PAGE check無表現
: BL21(DE3)pLysS 在induction後會繼續長，但SDS-PAGE check無表現
: western check在uninduce及induce都有表現但很少,leakage?
: Rosetta(DE3) transformation後就沒出現colonies了 (試過兩次)
: 我自己的推測是
: 此protein對E.coli有強毒性，所以一但induction菌就會掛掉或不生長
這個會部份表現在DT上。你沒測就說沒測就好了。
: 現在想到的是 1. 降溫induction延長時間
: 2. 換Rosetta(DE3)pLysS (但我們老師肯定不會買...太了解他orz)
: 3. 換表現菌種
: 4. 大量培養，硬是純化 (最後的辦法，算過大概需3 miligram)
蛋白多大？含多少positive charged residues？
另外如果你曾有染到（assuming CBB R-250）過這個蛋白，那麼SDS-PAGE這樣的
產量也許還是可以看到, or over-destain?
另外medium也有很大的差別。如果你在LB都養不出來，那我覺得你先去請人帶你
作一回比較快。
Transform以前不論的話：
Transform, did you use SOC for 30-60 min recovery?
Your competent cells are cultured by yourself or bought?
inoculation, Did you use single colony or multiple colonies?
Antibiotics storage condition?
(Minimal media) Adding vitamine complex and/or AA complex
(minimal media) How much Glc added?
what's the starting OD? (20mL, or 50mL into 1L) what's the final?
why did you use 0.4? You tested? paper? or former member?
Try small volume first to get the optimal condition or ask him/her.
Try auto-induction media
Try Celtone media etc
SDS-PAGE: boiled for sufficient time? reduced? different staining method?
for purified protein, how does the control/ STD look like?
...too many check points...
personal question: what's the last name of your professor? I may
recommend someone to help you.
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◆ From: 144.92.4.115
※ 編輯: renshiue 來自: 144.92.4.115 (05/11 07:40)