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Each Western blot experiment involves a unique antibody interacting with a unique sample under varying conditions. No single antibody or antigen concentration will work for every experiment. In order to obtain the most perfect Western blots, the antibody concentration needs to be optimized. The ideal antibody concentration is dependent on the concentration of antigen, the specificity and affinity of the antibody, and experimental conditions such as buffer composition.
Suboptimal antibody concentration can cause several common errors, including weak signal, nonspecific bands, and flecked or blotched background. Optimizing antibody concentration can resolve these problems and more, but performing multiple Western blots to obtain the optimal concentration is time consuming and wasteful. An easier, quicker, and cheaper method is to use a dot blot assay instead.
Dot Blot Protocol
Keywords: Western blotting optimization, antibody concentration, dot blot assay, best antibody concentrationClick for more optimization tips