|
|
★ ★ ★ ★ ★
西门吹雪170: 金币+5, 鼓励回帖交流 2014-08-20 16:49:11
有人还是先去看看免疫共沉淀的实验的原理吧！所要研究的可能存在相互作用的两种蛋白质——蛋白质X和蛋白质Y的彼此如果发生了相互作用是发生在细胞尚未裂解时！！！而后是抗其中一个蛋白质的单抗与其中一个蛋白质发生免疫识别！这步能够使蛋白质变性吗！！？？变性了还免疫识别个鬼呀！除非免疫簇就是序列免疫簇。序列免疫簇也是WB的实验基础原理之一，但是也是WB的局限性，WB不能检测出空间抗原免疫簇因为热煮沸和SDS变性而被破坏了的蛋白质！
免疫共沉淀的实验流程和简图如下：
“The procedure for immunoprecipitation is outlined in Fig. 1. In the first step, cells are transfected with plasmids coding for a bait protein and its potential ligand, the target protein. These cells are lysed with a gentle detergenttreatment, creating a lysate containing bait–target complexes along with many irrelevant proteins. A capture antibody that specifically recognizes the bait protein is added to the lysates, forming a new antibody–bait–target complex. The antibody is then used as a handle to immobilize the proteins on inert Sepharose beads that are covalently coupled to Protein A, which stably binds the constant regions of many types of antibodies. At this point, those proteins not immobilized on beads are removed by a series of washes. Finally, the bait-protein complexes are eluted from the beads and dissociated by boiling in SDS. The presence or absence of the target protein, which is the endpoint of the assay, is evaluated by Western blot.”
——————From : Haian Fu edits ,Protein-Protein Interactions:Methods and Applications,Humana Press,2004，338.
Haian Fu edits ,Protein-Protein Interactions:Methods and Applications,Humana Press,2004，
http://muchong.com/bbs/viewthread.php?tid=7764403
![蛋白质相互作用研究 蛋白A能pull-down蛋白B 反之检测不出]()
图1.JPG
|
|
回复此楼