Some preparations contain sodium azide as a preservative although it should be noted that azide can inhibit horseradish peroxidase, the most commonly used secondary antibody conjugate in western blots. The most commonly used blocking solution is a solution of 5% non-fat dry milk in PBS-T which works well for the vast majority of applications at a inexpensive price. Comparison between commonly used blocking buffers in western blotting. Contains immunoglobulins and serum proteins that can cross-react with the primary or secondary antibody.Incompatible with some anti-immunoglobulin antibody detection.Different sized proteins therefore require different formulations of acrylamide gel to get optimum separation (figure 1). Polyacrylamide gels are a matrix of cross-linked acrylamide monomers with the tightness of the mesh dependent upon the amount of acrylamide and cross-linker present. In a SDS page all proteins are denatured and have a uniform negative charge applied to them therefore migration is due to differences in size. What loading controls should be included to account for variability in loading and transfer ( see section 2.3)Ģ.1 Choosing an acrylamide gel percentage 2.1.1 Single concentration gels. What blocking solution to use for the experimental conditions ( see section 2.2).What percentage acrylamide gel to use in order to get the best separation ( see section 2.1).Proteins are separated by their mass before being transferred onto a membrane (either PDVF or nitrocellulose) and probed with antibodies to reveal expression of specific proteins.īefore starting any western blot experiment it is important to consider the following points: Proteins are first denatured before being loaded onto an acrylamide gel with an electric current applied. Western blotting, also known as immunoblotting, is a key technique in molecular biology to investigate changes in protein expression in a range of different tissue types. 2.1 Choosing an acrylamide gel percentageĢ.4 Selecting appropriate loading controlsĥ.1 Measuring the molecular weight of a proteinĥ.2 Quantifying protein expression from an immunoblot
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