![]() ![]() Although the indirect method requires one more wash and incubation step, it presents numerous advantages over the direct method, including amplification of signal and flexibility.įigure 1: (A) Directly conjugated primary antibody binds antigen bound to membrane. Multiple secondary antibodies bind to epitopes on the primary antibody, creating a labeled antigen-antibody complex (Figure 1(C)). After washing, the blot is incubated with a secondary antibody conjugated with the reporter enzyme or fluorophore. An unlabeled primary antibody forms a complex with the antigen bound to the blot membrane. The two step, indirect detection method of Western blotting avoids such interference with antigen detection. Excess primary may compensate for this effect but may lead to poor reproducibility and increased background. Figure 1(B) illustrates one of the problems of directly conjugating the primary antibody, whereby the reporter molecule can occlude the antigen binding region of the primary antibody, preventing good recognition of the antigen and leading to reduced sensitivity. Although this one step method is quicker, it is not widely used. In the direct detection method (Figure 1 (A)), a primary antibody directly conjugated to a reporter enzyme or fluorescent dye is used to detect the protein antigen on the blotting membrane after a single incubation step. Once proteins have been separated by gel electrophoresis and transferred onto a blotting membrane, Western blotting can be performed in either one or two steps. ![]() Each method can offer advantages depending on the experimental requirements. ![]() The technique can be performed either directly or indirectly. Download PDF Western blotting is a robust technique employing antibodies to detect proteins immobilized on a blotting membrane after separation by electrophoresis. ![]()
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