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果果gxl2013金虫 (小有名气)
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HRP与Peroxidase有区别吗？
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各位，请问有人知道HRP与Peroxidase有区别吗？新买的二抗写着：Peroxidase-conjugated affinipure goat anti-rat IgG(H+L)；我买的一抗官网说明写着：Rat Anti-Mouse LDL R Monoclonal Antibody (Catalog # MAB2255) followed by HRP-conjugated Anti-Rat IgG Secondary Antibody (Catalog # HAF005)。我做的从没出过条带，但是有背景。求大神解答 |
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2楼2017-05-26 13:41:04
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小木虫: 金币+0.5, 给个红包，谢谢回帖
果果gxl2013: 金币+1 2017-06-03 13:37:23
小木虫: 金币+0.5, 给个红包，谢谢回帖
果果gxl2013: 金币+1 2017-06-03 13:37:23
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????, ?????????^??????~ ????? SDS loading buffer ?c DTT ??????I??~ ????DTT ???????? DTT 250mM or 2-mercaptoethanol(?????F???е??) 12.5% (v/v) /5X sds loading buffer https://www.scientistsolutions.c ... mples#opennewwindow, ??????: Hello all, A co-worker of mine and I are debating the most efficient way of making our protein samples for Western Blots. She is convinced that heating at 50-70C for 10 minutes w/SDS is sufficient. I am convinced that samples need to be heated to at least 95C for 5 minutes w/SDS before proper denaturation can occur. The only reason I (personally) would use a slightly lower temperature is to avoid aggregation of multi-pass membrane proteins. Does anyone have any insight on this so we can settle our dispute? Thank you! kgambles Hi kgambles, Remember that your protein solution contains SDS and likely other denaturants, so there really is no need to heat the samples as high as 95oC. The combination of 70oC and denaturants for 10 minutes should be enough to denature a reasonably concentrated protein solution. Ivan Delgado ????o????????Y?υ???, ???sample protein ??????, loading buffer??SDS?????????. SDS is a detergent that is present in the SDS-PAGE sample buffer where, along with a bit of boiling, and a reducing agent (normally DTT or B-ME to break down protein-protein disulphide bonds), it disrupts the tertiary structure of proteins. This brings the folded proteins down to linear molecules. SDS also coats the protein with a uniform negative charge, which masks the intrinsic charges on the R-groups. SDS binds fairly uniformly to the linear proteins (around 1.4g SDS/ 1g protein), meaning that the charge of the protein is now approximately proportional to its molecular weight. |
10楼2017-06-02 18:50:24
3楼2017-05-26 13:41:54
果果gxl2013
金虫 (小有名气)
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4楼2017-05-26 15:19:48
果果gxl2013
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5楼2017-05-26 15:20:38
6楼2017-05-26 18:48:34
果果gxl2013
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7楼2017-05-26 21:12:59
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小木虫: 金币+0.5, 给个红包，谢谢回帖
果果gxl2013: 金币+3 2017-06-02 15:08:07
小木虫: 金币+0.5, 给个红包，谢谢回帖
果果gxl2013: 金币+3 2017-06-02 15:08:07
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我們小鼠肝組織, 也有用RIPA裂解, 然后12000 4度离心20分钟，取上清定量加LDS sample(loading) buffer+DTT(SDS sample buffer也可以)，70度10miin後冰上，-20度保存的。 https://www.scientistsolutions.c ... aking-wblot-samples |
8楼2017-05-31 14:09:20
果果gxl2013
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多有打扰，请多包涵9楼2017-06-02 15:11:44