Researchers rely on Western blotting (also called immunoblotting) to detect and compare target proteins in complex samples. This paper describes important considerations, strengths, and limitations of commonly used loading controls and normalization methods, to assist you in choosing a reliable method for your experimental context. Alternative approaches to Western blot normalization are examined, and the effects of normalization strategy on resulting data are explored. Important considerations for using a single internal loading control protein are discussed, as well as emerging alternatives like total protein staining that use aggregate measurements for normalization of sample loading.
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