※ 引述《ntutmpmpm (睡著了??)》之銘言： : sample以細菌細胞(E.coli)為主 : sample preparation的問題 在做TCA沉澱 加完lysis buffer即lysosome後 : 回溶率真的超級低的 放了頗久的時間pellet還是好大一塊 : 不曉得有經驗的前輩們是用什麼方式來解決回溶問題呢?? 雖然是很久以前的問題.... 不過最近的實驗遇到相同的瓶頸.... 唯一不同的是使用的來源是CHOA (mammalian cell) 收下細胞後 會先通一個Sepharose 4B column將thiol group protein elute出 然後再接TCA沉澱 沉澱完就遇到相同的問題: pellet很難完全溶解 因此必須找方法 將沉澱之後用2D sample buffer回溶的protein extract進行定量 我們實驗室用的protein quantification方法是 Bradford法 (Bio-Rad protein assay) 但是實驗室的過來人說 用這種方法定量 若是sample中含有urea則會使background太高 因此必須找別的方法來定量 或者是以別的濃縮方法來取代TCA沉澱 不過..... 參考相關paper 發現明明也有人用同樣的方法 (TCA沉澱 再用含有urea的buffer回溶 後接Bio-Rad assay) 以下是paper裡提到的methods: The protein pellets from the three protocols described above were washed with ice-cold methanol followed by multiple ice-cold acetone washes, dried in a flow of nitrogen gas and redissolved in IEF buffer (0.7 M urea, 2.0 M thiourea, 4% CHAPS, 10 mM DTT, 1.0% w/v carrier ampholytes pH 4–7 (Bio-Rad, Hercules, CA, USA)). Protein concentrations were quantified using the Bio-Rad protein assay (Bio-Rad) using BSA as a standard. We note that the BSA concentration is overestimated by approximately 50% using this reagent, resulting in an equivalent reduction in the protein loading of our samples (see manufacturer’s handbook); however, this approach allowed normalization of sample loading. 很冗長的問了一堆........>"""< 真是sorry拉 辛苦了大家的眼睛 想要知道 是不是有人也遇過跟我們一樣的問題呢...? 能不能請教一下高手們是如何解決這個瓶頸的... 是要相信paper裡說的 雖然Bio-Rad assay會有誤差 但還是照樣測 還是還有更好的方法.......?? 或者是有更好用的precipitation / protein quantification方法????? -- ...And she was richer in those dreams than in realities; for things seen pass away, but the things that are unseen are eternal. --

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