Western Blot

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is an electrophoresis method commonly used to separate proteins based by size. SDS is an anionic detergent, a detergent that has a negative charge, that binds to proteins and disrupts the non-covalent forces that are responsible for their three-dimensional structure, thus denaturing the proteins. SDS also gives the proteins a uniform net negative charge that is relative to its size. This causes the denatured proteins to migrate through the gel during electrophoresis solely based on its size. People typically do SDS-PAGE prior to Western blotting. SDS-PAGE separates the proteins while Western blotting involves the transfer of proteins from the gel to a membrane, and confirmation of the presence/absence/expression level of the proteins using an antibody.

Native or non-denaturing gels do not use SDS. Nor is a reducing agent, such as DTT or beta-mercaptoethanol, added to the sample loading buffer. This means that the proteins maintain their native structure and charge. As a result, they will migrate through the gel during electrophoresis according to both their mass and their charge. Native gels are used to determine the aggregation state of a protein, study protein complexes, and isolate enzymes.

The amount of protein that should be loaded into the loading gel for a Western blot depends a several factors including the expression level of the protein and the sample being probed. As a rule of thumb, 20-30 µg of total protein is loaded per well for cell and nuclear lysates and membrane samples. Ten to 100 ng of protein is typically loaded if probing for a purified protein. The appropriate protein amount to load, though, should be determined prior to starting a Western blot experiment.

Proper controls for Western blotting are important for determining the source of problems and for validating results. There are several controls that need to be used for a proper Western blot experiment.  

1. Positive Control Lysates – Positive control lysates are from cell lines or tissue samples that are known to express the protein of interest. This control will yield a positive band on the Western blot. 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.

2. Negative Control Lysates – Negative control lysates are from samples known to not express the target protein. This control will yield no band on the Western blot. This control is important for determining non-specific binding of the antibodies.

3. Positive Endogenous Control Lysates - Positive endogenous control lysates are from samples that are known to express the target of interest. This control should be used when testing a sample of recombinant protein, such as a tagged protein. Folding of a recombinant protein may be different than the native protein, and misfolding may prevent the antibody from accessing the epitope. 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.

4. Loading Control – Loading controls are housekeeping proteins, which are proteins that are expressed at equivalent levels in almost all tissues and cells. This control ensures that differences in protein expression between wells isn’t due to loading or protein transfer errors. Loading controls are required for the semi-quantification of protein levels between wells.

Simple Western assays are automated Western blot. This platform automates all the steps involved in electrophoresis and Western blotting, from protein loading and separation, immunoprobing, washing, detection and quantitative analysis of data. View the Simple Western FAQs page on the Novus Biologicals website for more information on Simple Western.