![]() To determine the appropriate amount of lysate to load on the gel, load decreasing dilutions of lysate on the gel. If too much lysate is loaded onto a gel, antibodies can bind non-specifically to proteins of excessive abundance, resulting in multiple bands. If multiple bands are still observed, then the secondary antibody is responsible for the artifacts. To determine quickly whether the primary antibody is responsible for producing multiple bands on the blot, perform the entire Western blot procedure but omit the primary antibody. Both antibodies can be titrated simultaneously using a dot blot in a checkerboard like fashion. To determine if high antibody concentrations are resulting in multiple bands, titrate the antibodies. Non-specific binding can occur with any protein, thus bands of higher and lower molecular weight may be observed. If the concentration of either the primary or secondary antibody is too high, the antibody can bind non-specifically to proteins other than the protein of interest. The types of bands that are observed can help determine the cause of the artifact.Ĭoncentration of Primary or Secondary Antibody too High Technical artifacts can cause the appearance of bands that have higher or lower molecular weights than the actual protein. Multiple Bands Caused by Technical Artifacts This guide describes various causes of multiple bands due to technical artifacts and how to determine if multiple bands represent true variants of the protein of interest. When dealing with multiple bands on Western blots, it is important to determine whether they are due to technical artifacts or whether they represent true variants of the protein of interest. However, analysis of the protein can be difficult if multiple bands appear on the blot. The Western blot assay provides valuable information about a protein including abundance, the apparent molecular mass, post-translational modifications and splice variants.
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