WesternBlotTechnique.ppt
《WesternBlotTechnique.ppt》由会员分享,可在线阅读,更多相关《WesternBlotTechnique.ppt(21页珍藏版)》请在三一文库上搜索。
1、Western Blot Technique,speaker:,catalogue,1.History Of Western Blot 2.Principle Of Western Blot 3.Process Of Western Blot 4.Common Questions & Solutions,1.History Of Western Blot,1).Biological macromolecular blotting method was first put forward by E.M.Southern in 1975, who studyed at the university
2、 of Edinburgh , Scotland. 2).In 1979, Towbin et al. first picked up antigen, using electroelution (电洗脱)device as a blotting force. And called it immunoblotting, Corresponding to the Southern (DNA molecule hybridization), Northern (RNA molecule hybridization). 3).In 1981, Burnette named Western blott
3、ing to repalce immunoblotting as protein imprinting method.,2.Principle Of Western Blot,The process of Western Blot is similar to Southern or Northern blot, While Western Blot uses PAGE to isolate protein and its detecting object is protein. Its “probe“ is antibody. The technology is widely used in
4、detecting the expression of protein levels.,Protein samples, after separated via PAGE, is transferred to the solid carrier (such as nitric acid cellulose film), which can adsorb protein in the form of non covalent bond and keep peptides activities. Protein or peptide is regarded as antigen, then exe
5、rts a immune response with antibody.Next, there is a reaction related with primary antibody and second antibody marked with enzyme or isotope(同位素).After the Chromogenic substrate(底物显色) or autoradiograph(放射自显影) from imaging, we can detect the protein and the expression of target gene.,3.Process Of We
6、stern Blot,electrophoresis and embrane transfer,blocking,primary antibody,secondary antibody,washing,detecting,1)electrophoresis,3.1.1Choice SDS-PAGE:protein with a single subunits Native-PAGE(非变性):protein with more than one subunits electrophoresis in Tris-Tricine:peptide or protein with MW less th
7、an 10kD 3.1.2SDS-PAGE,SDS is an anionic detergent (阴离子去垢剂) Amino acid side-chains is bond with SDS, forming a SDS -protein compound with negative charge.The number of negative charge adsorbing on the surface of compund is considerably more than the original charge, eliminating the original charge di
8、fference between protein molecules.,Attention:Polyacrylamide has neurotoxicity, wear gloves when operating,2)membrane transfering,3.2.1.Membrane Choosing Nitrocellulose(硝酸纤维素,NC)membrane Polyvinylidene Fluoride(聚偏二氟乙烯, PVDF)membrane Selection Standards: A.The combine-ability with protein molecules B
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- WesternBlotTechnique
链接地址:https://www.31doc.com/p-2977529.html