Protein Identification by Western Blot

Western blotting uses antibodies to identify proteins after the proteins have been separated by gel electrophoresis. In order to make the proteins accessible to antibody detection, they are moved from within the gel onto a membrane composed of nitrocellulose or polyvinylidene difluoride (PVDF). In a basic transfer protocol, the membrane is placed on top of the gel, and the assembly is sandwiched between stacks of filter papers. The gel side of the stack is placed in a buffer solution which moves up the paper by capillary action, through the stack, bringing the proteins with it. The proteins are carried through the gel and bind to the transfer membrane.

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Another method for transferring the proteins is called electrophoretic blotting (or electroblotting) and uses an electric current to pull proteins from the gel onto the blotting membrane. With either method, the proteins move from within the gel onto the membrane while maintaining the spatial separation they had within the gel. Once transferred to the membrane the proteins are accessible for detection.

Methods of transfer include wet, semi-wet, semi-dry, and dry blotting. Semi-dry blotting can be performed with the Novex Semi-Dry Blotter. Dry blotting can be performed with the iBlot Gel Transfer Device. Semi-wet blotting can be performed with the XCell II™ Blot Module. Transfer efficiency can be checked using reversible membrane stains. After the transfer, the membrane is probed for the presence of specific proteins using primary antibodies. The probing of the membrane with antibodies can be conducted manually, or it can be automated using the BenchPro 4100. Figure 1 outlines the western blotting workflow. The presence of the target protein(s) is typically visualized using chromogenic, chemiluminescent, or fluorescent detection reagents (i.e., goat anti-rabbit HRP).

The iBlot Western Blotting System