※ 引述《ntutmpmpm (睡着了??)》之铭言： : sample以细菌细胞(E.coli)为主 : sample preparation的问题 在做TCA沉淀 加完lysis buffer即lysosome後 : 回溶率真的超级低的 放了颇久的时间pellet还是好大一块 : 不晓得有经验的前辈们是用什麽方式来解决回溶问题呢?? 虽然是很久以前的问题.... 不过最近的实验遇到相同的瓶颈.... 唯一不同的是使用的来源是CHOA (mammalian cell) 收下细胞後 会先通一个Sepharose 4B column将thiol group protein elute出 然後再接TCA沉淀 沉淀完就遇到相同的问题: pellet很难完全溶解 因此必须找方法 将沉淀之後用2D sample buffer回溶的protein extract进行定量 我们实验室用的protein quantification方法是 Bradford法 (Bio-Rad protein assay) 但是实验室的过来人说 用这种方法定量 若是sample中含有urea则会使background太高 因此必须找别的方法来定量 或者是以别的浓缩方法来取代TCA沉淀 不过..... 参考相关paper 发现明明也有人用同样的方法 (TCA沉淀 再用含有urea的buffer回溶 後接Bio-Rad assay) 以下是paper里提到的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. 很冗长的问了一堆........>"""< 真是sorry拉 辛苦了大家的眼睛 想要知道 是不是有人也遇过跟我们一样的问题呢...? 能不能请教一下高手们是如何解决这个瓶颈的... 是要相信paper里说的 虽然Bio-Rad assay会有误差 但还是照样测 还是还有更好的方法.......?? 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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