The membrane is then incubated with a primary antibody that specifically binds the protein of interest.įollowing a further blocking step, a conjugated secondary antibody binds the primary antibody allowing the target protein to be visualized.Įither colorimetric or chemiluminescent substrates can be used to visualize signal from reporter enzyme conjugates. Protein transfer is followed by a blocking step, which uses a protein solution to minimize non-specific interactions. Following separation, the proteins are transferred (electroblotted) onto a membrane by placing the gel and membrane in direct contact and applying an electric field to pull the proteins into the matrix. The density of the gel retards their migration, allowing them to be separated by size. The proteins migrate towards the positive electrode. The sample is then loaded into the gel, and a voltage is applied. Sodium dodecyl sulfate-polyacrylamide denaturing gel electrophoresis (SDS-PAGE) uses SDS to coat the proteins, masking their intrinsic charge and imparting an overall uniform charge proportional to their size. Protein extraction may be followed by denaturation and reduction to convert the proteins to their primary structure, which allows them to be separated by their molecular weight during electrophoresis. For example, when screening for expression, proteins are extracted by lysing cells to release the proteins, so they can enter the separation matrix. Recombinant proteins from expression systems and endogenous proteins from biological samples can all be detected with western blot.įor proteins to be analyzed by western blot, the sample containing them must first be processed to make the target proteins accessible for analysis. Western blotting can be used to detect proteins from both natural and synthetic sources. Protein transfer (electroblotting) onto a membrane.Protein separation by gel electrophoresis.Western blotting is performed in six stages: This technique provides information about the target protein(s), including abundance and molecular weight, and provides a way of detecting protein-protein interactions and post-translational modifications. Western blotting is widely used today in molecular biology, biochemistry and cell biology to detect specific proteins of interest from biological samples.
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