After separation by electrophoresis, proteins can be bound to membranes where they are fixed and readily accessible for immunological or biochemical analyses, quantitative staining, or the identification of protein-protein and protein-ligand interactions. Western blotting, the transfer of proteins to a solid-phase membrane support followed by immunodetection, is a powerful and popular technique for the visualization and identification of proteins. Western blotting combines the resolution of gel electrophoresis with the specificity of immunoassays, allowing individual proteins in mixtures to be identified and analyzed. This section provides an overview of western blotting methods, equipment, membranes, transfer buffers, transfer conditions, detection, and imaging.
A special section, the Western Blot Doctor™, is a self-help guide developed by Bio-Rad researchers that enables you to identify and troubleshoot western blotting problems. Comprehensive solutions and suggestions are provided to help solve your particular western blotting challenges.
The most commonly used protein blotting technique, western blotting (immunoblotting), was developed to probe for proteins that were inaccessible to antibodies while in polyacrylamide gels. Western blotting refers specifically to the immunological detection of proteins that have been separated by gel electrophoresis and transferred onto a membrane. Since the development of immunoblotting techniques, other probing and detection techniques have been developed for functional protein characterization (for a review, see Kurien and Scofield 2003).
The western blotting workflow involves two phases: protein transfer to a membrane and detection of the membrane-immobilized protein.
Protein Transfer to a Membrane
The first phase of western blotting is the transfer step, which consists of moving the proteins from a solution or gel matrix to a synthetic membrane support where it is bound, forming the blot. Proteins can be transferred to membranes using a number of methods, but the most common are electrophoretic transfer (electroblotting) and microfiltration (dot blotting). In general, electrophoretic transfer is used to transfer proteins following electrophoretic separation by native or SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and microfiltration is used to transfer proteins that are in solution. Capillary blotting methods, such as those used for nucleic acid transfer, are seldom used to transfer proteins from gels.
Protein Detection on the Membrane
The second phase, protein detection, entails probing the membrane with either a protein stain or antibodies specific to the protein of interest, and subsequent visualization of the labeled proteins. Western blot detection involves a number of steps, including selection of the appropriate protein detection method, blotting buffers and reagents, and gel and blot imaging equipment.
The protein blotting workflow involves selection of the appropriate method, apparatus, membrane, buffer, and transfer conditions. Once proteins are immobilized on a membrane, they are available for visualization, detection, and analysis.
Kurien BT and Scofield RH (2003). Protein blotting: a review. J Immunol Methods 274, 1–15.
Jensen CE (2012). The basics of western blotting. Anatomical Record 295(3) 369–371.