Western blotting is a well-established analytical technique for detecting, analyzing, and quantifying proteins. This method is widely used to detect specific protein molecules in complex samples such as tissue homogenates and cell lysates. Western blotting typically involves protein separation by gel electrophoresis followed by transfer to a polyvinylidene difluoride (PVDF) or nitrocellulose membrane. After proteins have been transferred, they can be stained for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection.
In Western blotting immunodetection, proteins are identified through their binding to specific antibodies. Typically, a primary antibody is used in combination with an HRP- or AP-conjugated secondary antibody for chemiluminescent or colorimetric detection using an appropriate substrate. Alternatively, a fluorescently labeled primary or secondary antibody can be used for direct visualization.
Western blotting is used extensively in biochemistry to detect the presence of specific proteins, to determine the extent of post-translational modifications, to verify protein expression in cloning applications, to analyze protein and biomarker expression levels, in antibody epitope mapping, and to test for markers of disease in clinical settings.
The need to simultaneously analyze more proteins in limited samples has driven ongoing research into improving the sensitivity and speed of blotting techniques. Double blotting eliminates false positives caused by nonspecific interactions. Far-Western blotting enables the detection of specific protein-protein interactions. Southwestern blotting is used to identify proteins that interact with specific DNA sequences. Multistrip blotting increases throughput while minimizing inter-blot variability. New technologies are being developed to reduce the amounts of protein required to produce a signal and improve the quantitative capabilities of Western blotting.
When working with cells or tissues, samples must be disrupted to isolate the proteins prior to analysis. Lysis and extraction buffers use detergents to break down cell walls and membranes to release the proteins. The optimal buffer should be chosen based on the sample type and sub-cellular localization of the target protein.
Protein gel electrophoresis is used to separate and resolve proteins prior to blotting. The prepared protein mixture is run on a polyacrylamide gel to sort proteins by molecular weight and charge.
Target protein is detected using a primary antibody in combination with an HRP- or AP-conjugated secondary antibody and an appropriate chemiluminescent or colorimetric substrate, or by using fluorescently-labeled primary or secondary antibody.