蛋白组学试验中的常见问题及解答1关于使用三氯醋酸-中国试剂网.PDF

中国试剂网 3.15.3.26 蛋白组学实验中的常见问题及解答 1.关于使用三氯醋酸沉淀法后蛋白质难于重悬的问题 Q:1,Hi !I have been trying to clean and concentrate proteins form eucariotic cell culture medium in order to analyse them in 2D. I’ve read some papers were TCA precipitation was used and recommended for this purpose but I have not been able to redissolve my samples. I have used 10%TCA and let it precipitate for 30 minutes in Ice, afterwards I have centrifuge the sample, washed it with diethil ether and tried to dissolve the pellet in ordinary IEF sample solution containing Urea, Chaps, ampholites and DTT. I wasn’t able to dissolve the sample even using a 9M Urea concentration. Do you have any suggestions on how to dissolve proteins from TCA precipitation or an alternative way to clean and concentrate proteins from culture medium for 2d analysis? Thank you! A:This problem occurs very often. Better for cleaning and concentration of protein samples: Use the Ettan 2-D Cleanup Kit from Amersham Biosciences 点评:这个问题由AMERSHAM 著名科学家回答, 比较会替公司做广告. 其实真核细胞总蛋白质的提取用不着使用 TCA/acetone 法,细胞相对于植物组织 还是比较干净的,少掉了一些脂类\酚类杂质.对于组织来讲,使用clean-up kit 效果 还是不错的 2.在第二向电泳中,为什么我的溴氛蓝条带都跑到玻璃板下缘了而蛋白质只跑了 一半啊,郁闷! Q:Can anyone help with a recent problem with proteins only running half way despite the running dye reaching the end. This problem has suddenly appeared, running buffer is new batch but has been pHd and made to the same recipe as before. Gels are 10%T being run under the same conditions as always (70V overnight at RT). Not only do the proteins on the gel seem to disappear towards the bottom half but the molecular weight markers also become very faint and blurry. It almost looks as if the low Mw