Western Blot Technique.pptVIP

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Western Blot Technique

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 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 blotting 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 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 exerts 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 Western Blot 1)electrophoresis 3.1.1Choice SDS:protein with a single subunits Native(非变性):protein with more than one subunits electrophoresis in Tris-Tricine:peptide or protein with MW less than 10kD 3.1.2SDSSDS 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 o

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