Our partner GeneTex supports researchers with a comprehensive selection of troubleshooting tips for many issues which can occur in a western blot (WB) experiment.ĭownload also the Western Blot Troubleshhoting Guide from GeneTex as pdf.īe sure to load proper positive and negative controls to ensure that the WB procedure is performed correctly.Ī. Loading controls are required for the semi-quantification of protein levels between wells.A comprehensive selection of troubleshooting tips This control ensures that differences in protein expression between wells isn’t due to loading or protein transfer errors. Loading Control – Loading controls are housekeeping proteins, which are proteins that are expressed at equivalent levels in almost all tissues and cells. This control will let the researcher know that a negative result is due to the epitope being blocked as opposed to the protocol not working.Ĥ. Folding of a recombinant protein may be different than the native protein, and misfolding may prevent the antibody from accessing the epitope. This control should be used when testing a sample of recombinant protein, such as a tagged protein. Positive Endogenous Control Lysates - Positive endogenous control lysates are from samples that are known to express the target of interest. This control is important for determining non-specific binding of the antibodies.ģ. This control will yield no band on the Western blot. Negative Control Lysates – Negative control lysates are from samples known to not express the target protein. This control is important as it ensures there were no issues in the Western blot protocols and that any negative results are due to a lack of protein in the sample and not to procedural problems.Ģ. This control will yield a positive band on the Western blot. Positive Control Lysates – Positive control lysates are from cell lines or tissue samples that are known to express the protein of interest. There are several controls that need to be used for a proper Western blot experiment.ġ. Proper controls for Western blotting are important for determining the source of problems and for validating results. A band will appear at approximately its molecular weight, as marked by the molecular weight ladder. Secondary antibodies bound to enzymes or fluorochromes are applied to the membrane to visualize the protein/antibody complex. The membrane can be incubated with primary antibodies specific for target protein(s) of interest. The proteins are then transferred to a membrane by application of an electrical current. This causes the proteins to separate by size, with the smaller proteins traveling faster through the gel. The proteins pass from the stacking gel into the separating gel, which is more basic and has a higher gel concentration. This environment allows the proteins in the sample to form highly defined, sharp bands. The stacking gel contains less acrylamide and has a lower pH. Polyacrylamide gels that are used consist of two sections: a stacking gel and a separating gel. In Western blot, samples are loaded onto a polyacrylamide gel and separated based on their molecular weight by SDS-PAGE electrophoresis.
Due to the specific nature of antibody binding, Western blot analysis can be used to detect and quantify a single protein within a mixture of thousands of different proteins. Western blot is a laboratory method that uses antibodies to identify individual proteins in cell or tissue lysates.
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