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国外蛋白组学专业BBS的一些问答-强烈推荐（11-14更新） [精华]

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这个帖子发布于16年零18天前，其中的信息可能已发生改变或有所发展。

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
codegreen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,等过几天做实验了,小弟把自己做的使用clean-up kit前后的2-D图谱之对比贴出来.

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2003-11-01 21:22 浏览 : 6080 回复 : 18

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codegreen 编辑于 2003-11-15 01:34

• 丁香早知道 - 11.15 | 想离开医院去当老师，我爸就跟我断绝父子关系！

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codegreen

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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 pH'd 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 proteins have somehow diffused out of the gel, although this does not seem possible. I know that I cannot expect low Mw proteins to focus as sharply as high, however they have never been this fuzzy before.
Thanks
A:Never "pH" the running buffer!! In this way you have introduced Cl- Ions into the Tris-glycine buffer: this is a No-No. The system has to move all Cl-ions out of the cathodal buffer tank before glycine and proteins start to migrate. When you make your running buffer, just use Tris, glycine and SDS of good quality, according to the stsndard recipe. You do not need to measure the pH; and you must not titrate the running buffer!
codegreen点评:兄弟们知道这个同志犯了什么低级错误吗,他老人家自做聪明使用HCL去调Tris-Glysin running buffer的PH值,危害是电泳系统在开始迁移甘氨酸和蛋白质之前必须把阴极的CL离子都跑出去,结果自然就造成了蛋白质斑点迁移的滞后了,呵呵.

2003-11-01 21:43

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codegreen 编辑于 2003-11-03 22:39

• 丁香早知道 - 11.13 | 母亲急性心梗，我给母亲做了人生第一台急诊手术！

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codegreen

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今天已经申请协助主任管理蛋白组学专栏，俗话说新马仔上任三把火，偶第一天上来不能弱了主任的名头，所以想了一个新花样，以后还会继续选有代表性的问题（troubleshooting)上来，解决大家的常见问题。其实小弟也是半罐水响叮当，希望能以自己的顽石引出高手们的美玉。大家请踊跃参加讨论，如果有兄弟能回答得有道理，小弟一定建议主任加分！

2003-11-01 21:53

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codegreen 编辑于 2003-11-01 21:59

• 一份心梗的心电图让我犹豫了，求指导。

kiwi

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能不能贴一下原文的网址啊？thank ul

2003-11-03 19:20

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• 丁香热议——硕士毕业三甲去不了，二甲不想去，民营是最好选择？

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