It offers the following specific advantages (a) wet membranes are pliable and are easy to handle compared to gels, (b) easy accessibility of the proteins immobilized on the membrane to different ligands, (c) only small amount of reagents are required for transfer analysis (d) multiple replicas of a gel are possible (e) prolonged storage of transferred patterns, prior to use, becomes possible and (f) the same protein transfer can be used for multiple successive analyses ( 7– 9). This procedure ( 1, 2) is a powerful tool to detect and characterize a multitude of proteins, especially those proteins that are of low abundance. Protein transfer with subsequent immunodetection has found wide application in the fields of life sciences and biochemistry. The utility of the high resolving power of SDS PAGE ( 5) was limited in purpose, owing to the fact that the separated proteins in the gel matrix were difficult to access with molecular probes, until the advent of protein blotting. (C) Primary antibody binding to a specific band on the blot (D) Secondary antibody conjugated to an enzyme (alkaline phosphatase or horse radish peroxidase) binding to primary antibody (E) color development of specific band (Reproduced from Ref 10 with permission from Elsevier). The bands shown are hypothetical (B) Exact replica of SDS PAGE gel obtained as a blot following Western transfer. (A) Unstained SDS PAGE gel prior to Western blot. Schematic representation of Western blotting and detection procedure Dot blotting refers to the analysis of proteins applied directly to the membrane rather than after transfer from a gel. The subsequent employment of antibody probes directed against the membrane bound proteins (immunoblotting) has revolutionized the field of immunology ( Fig. The blotted proteins form an exact replica of the gel and have proved to be the starting step for a variety of experiments. The term ‘western blotting’ was coined to describe ( 5) this procedure to retain the ‘geographic’ naming tradition initiated by Southern’s paper ( 3) ( see Chapter 3). Protein blotting evolved from DNA (Southern) blotting ( 3) ( see Chapter 1) and RNA (Northern) blotting ( 4). Nucleic acids are routinely transferred from agarose gels, to a membrane support, through capillary action (Southern blotting). Proteins that are resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) gels are typically transferred to adsorbent membrane supports under the influence of an electric current in a procedure that is known as western blotting (WB) or protein blotting ( 1, 2) ( see Chapters 2 and 3). The transfer of proteins or nucleic acids to microporous membranes is referred to as “blotting” and this term encompasses both “spotting” (manual sample deposition) and transfer from planar gels.
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