 Western Blotting Procedure This is a procedure of western blotting. First of all, sample preparation, then gel electrophoresis. At third step, blotting or transfer. At fourth stage, there is blocking, then antibody probing and the last step is detection. How western blotting is performed? Usually the choice of extraction method depends primarily on the sample size and the type of the sample. Whether the analysis is targeting all proteins of a cell or only a component from a particular subcellular fraction. It depends on the procedure, whether someone is studying all the proteins which are present in a cell or the person is studying a small component of a protein. Samples are loaded into separate wells and protein marker is also loaded. The separated protein mixture are transferred to a solid spot for further analysis of the proteins. Transfer can be done in wet or semi-dry conditions. There are two type of the transfer methods in case of western blotting. It can be wet, it can be semi-dry conditions. Semi-dry transfer is generally faster. Wet transfer is recommended for large size proteins which are larger than 100 kilodalton. The fourth step is blocking. That is an important step in immunodetection phase of western blotting because it prevents non-specific binding of antibody to blotting membrane. Here we can see that how electrophoretic transfer is performed in case of western blotting. This is the diagram of gel electrophoresis in which we can see that this is the buffer tank. Here is the gel along with the filter papers. If we magnify this we can see that on the ends there is a sport grid. Here we can see that this is in black color. Then there are pads that can also be tissue papers. Then filter paper here. Transfer membrane that is in white color and gel that is in blue color. If we magnify this portion we can see that here is the gel. This is gel and here is the transfer membrane. The bands or the proteins which are present in the gel it will transfer on the membrane. So this is the diagram how the protein is transferred from gel on the membrane. Protein of interest is detected and localized using a specific antibody. Once the protein is transferred from the gel on the membrane then the protein is detected with the help of specific antibodies. Western blotting protocol utilize a non-label primary antibody then that is directed or checked against the target protein. A specific label secondary antibody is used that will bind to the primary antibody. The most common antibody label used in case of western blot is HRP. So once the protein is transferred from gel on the membrane then primary antibody is used. Then against the primary antibody secondary antibody is used and the secondary antibody that is labeled with the help of HRP. The signal is detected when HRP is exposed to substrate solution in the final step of the immunodetection procedure. Here we can see in this diagram that proteins on the membrane after transfer from gel these are the proteins which are transferred on the membrane. Now against these proteins primary antibody they are attached. So this is the primary antibody. Now against the primary antibody this is the secondary antibody. Secondary antibody is attached to the primary antibody and primary antibody will attach to specific protein in the mixture of the proteins. Secondary antibody is labeled with HRP. Detection regent reacts with HRP and generates light emission by which we can see that there is presence of particular protein in mixture of the proteins. Western blotting is a technique which is used to identify specific proteins in mixture of proteins.