![]() The appropriate concentration of protein in the sample is determined by a spectrophotometer.This process is carried out at a lower temperature in the presence of protease inhibitor to prevent the denaturation of proteins.Proteins present in the cell cytosol are extracted by mechanical, chemical or enzymatic lysis of cell.The most common form of sample for western blotting are the cell lysates.Step 1: Sample preparation or Cell lysis to extract protein: Western blotting technique involves the following steps:.A more sensitive way is the use of a chemiluminiscent compound with suitable enhancing agents to produce light at the protein site and make it visible.If enzyme-linked antibodies are employed, then addition of chromogenic substrate produces a highly colored and insoluble product at the site of target protein and makes it visible.If the protein of interest is bound by radioactive antibody, its position on the blot is determined X-ray autoradiography.The antigen-antibody complexes can be visualized in a variety of ways.The desired protein on the membrane can either be detected by the use of a labeled primary antibody directed against the protein or a labeled secondary antibody directed against the primary antibody in antigen-antibody complex.The protein bands are transferred to a nitrocellulose membrane by an electrophoretic process called blotting where the proteins are then immobilized.A protein mixture is electrophoretically separated on an SDS-polyacrylamide gel (SDS-PAGE), a slab gel infused with a dissociating agent called Sodium dodecyl sulphate (SDS).In western blotting, an individual protein of interest is identified using radiolabelled or enzyme-linked polyclonal or monoclonal antibody specific for that protein.You may also want to see: Southern Blotting: Principle, Steps involved and Applications It is similar to Southern blotting (that detects DNA fragments) and Northern blotting (that detects mRNAs).Western blotting technique is used for the separation and identification of a specific protein in a complex mixture of proteins based on their molecular weight. ![]()
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