Western blotting, or immunoblotting, is a widely used and powerful analytical tool used to detect specific proteins, or protein modifications, in a sample of tissue/cell homogenate or extract. There are numerous variations of western blotting equipment and reagents that allow for detection and measurement of a multitude of proteins with a wide array of molecular properties.
In 1979, Towbin, et al., introduced a new, previously unknown technique to separate and identify proteins, and called it western blotting1. This technique allows for the separation, detection and measurement (qualitative and quantitative) of specific targets, through antibody/antigen interactions, within a complex biological sample such as cell and/or tissue lysates. Western blotting, or immunoblotting, can be used to detect proteins and post-translational modifications (ex. phosphorylation) as well as confirm gene expression, and is now a mainstay of most research labs.
The first step in western blotting is to separate the proteins contained within the sample, which is done using gel electrophoresis. Biological samples are loaded into a gel and electricity is passed through allowing the separation of proteins by molecular weight. Molecular weight is inversely proportional to migration in a gel; therefore the smallest proteins travel the furthest down the gel. Protein “ladders” that contain dyed proteins can be loaded at either end of the gel to illustrate where specific molecular weights will travel down the gel to aid in the identification of the specific target of interest. The proteins contained within the gel are then transferred, or blotted, out of the gel and onto the surface of a membrane (ex. PVDF or nitrocellulose). To prevent non-specific antibody binding, the membrane is incubated in a protein blocking buffer, and then probed with a primary antibody specific to the protein of interest. After which, the membrane is incubated in a secondary antibody that is specific to the host species of the primary antibody. The secondary antibody is conjugated to a reporter molecule that allows the detection of the antibody signal. Between each incubation period the membrane is washed with a buffer containing a detergent to wash any non-bound antibodies from the surface of the membrane.
There are several varieties of reporter molecules and hence detection methods available. Colorimetric detection can be produced by using horseradish peroxidase or alkaline phosphatase conjugated to the secondary antibody. Colorimetric substrates produce a precipitate that is deposited on the membrane and is visible to the naked eye. Currently the most sensitive and popular detection method uses chemiluminescent substrates. The reaction with the enzyme conjugated to the secondary antibody produces light as a byproduct and can be captured with film. The light output can also be measured with digital imaging based on charge-coupled device (CCD) cameras, which are gaining in popularity as an alternative to film for capturing the signal. In addition, fluorescent tags can also be bound to secondary antibodies but require an instrument capable of capturing fluorescent signals. Western blotting using fluorescence is a somewhat newer technique that allows for some interesting potentials, including the possibility of multiplexing that would allow for the measurement of multiple targets on a single blot without stripping and re-incubating in antibodies. Whichever method of detection is used, the amount of protein on the blot should correlate with the intensity of the signal obtained.
Western blotting is a vigorous research tool that allows scientists to detect and, with specific methods, measure the amount of protein present in a sample. Experimentally, western blotting can be used to measure and compare expression of proteins, post-translational modifications and gene expression in control and experimental tissues or cell lysates. In the clinical setting, western blotting can also be used to examine differences in protein expressions between healthy and diseased tissues.
Bethyl Laboratories sells high quality Western blot antibodies. These products have recently been used to study:
Thousands of Bethyl antibodies are validated for use in western blotting. The complete list is here: