WesternBlotTechnique.ppt

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,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 than 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 difference 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.The size of membrane pore C. Whether affect the following color detection D. The other requirements of subsequent experiments,3.2.2.Method Choosing Wet transfer system,Half-dry transfer system,3)Blocking,Purpose To prevent primary antibody combining with membrane , the potential binding sites of the membrane should be blocked. Materials 5%non-fat milk or 3% BSA Condition room tempture, 2h or 4 ，overnight,4）incubating with antibody,NC membrane,incubating in culture dish,room tempture, 2h or 4 ，overnight,solution with primary antibody,washing with TBST,3-5times, 5-10min per time,solution with secondary antibody,incubating in culture dish,room tempture, 2h or 4 ，overnight,washing with TBST,3-5times, 5-10min per time,observing chromogenic substrate,Different methods in marking secondary antibody and imaging antibody labeled by 125I antibody labeled by fluorescence antibody labeled by enzyme（HRP/AP） Using enzyme substrates to produce color reaction or chemiluminescence reaction,X - ray tablet (X-光片压片显色),Bio-RAP (凝胶成像仪),4.Common Questions & Solutions,Q1:Glue is uneven, Gel electrolyte leaks A:Glue glass should be clean; Temperature should be appropriate; The bottom of two pieces of glass should be on a line; Reagent should be mixed up sufficiently Q2:Band is more narrow than the normals or not clear A:Gel polymer is not uniform(均匀）; The sample-well is bending; Samples have a high concentration; Protein concentration is too low;,Q3:The quantities of protein transferred to the membrane is small A: The MW of protein is less than 10kD The pI of protein is less than 9 The concentration of SDS is not appropriate The gel is too thick S:Reducing the time of transferring when the MW of protein is less than 10kD.Using the membrane with small pore Using the high pH Buffer Extending the time of transferring,Q4:The hybridization signal is very weak A:The antibody concentration is too low Antigen is not enough Transferring is inadequate Membrane is washed excessively The antibody is not suitable for western blot S: Increasing the concentration of antibody Increasing the amount of sample-protein Reducing the time and number of washing,Q5:There is a nonspecific band A:The primary antibody is not the only specific Secondary antibody is combined nonspecificly S: Reselecting antibodies Seting up a parallel comparison experiment to detect whether there is a nonspecific combination with secondary antibody,Thank You All！,SunnyRain,