Question:
How to detect protein using western blotting?
The Protein Man Says:
Western blotting is one of the most important techniques used in protein detection and identification. By using this technique, researchers can accurately detect the presence and isolate a particular protein of interest from a mixture of proteins present in any biological sample. In addition, Western blotting can also be used to estimate the size of a protein, confirm the presence of post-translational modifications and compare the levels of proteins between samples.
Western Blotting: An Overview of the Process
In doing a Western blot, individual proteins extracted from a biological sample and separated based on their molecular weights through gel electrophoresis. The protein of interest is then transferred to a membrane support and incubated with a primary antibody specific to the protein of interest. All unbound antibodies are then washed off and the bound antibodies are detected through one of the direct or indirect methods available.
Protein Detection: Some Commonly Used Methods
Generally, the protein of interest can be detected through chemiluminescence, fluorescence and colorimetric methods.
Chemiluminescence method
In detecting proteins through the chemiluminescence method, researchers commonly use HRP-conjugated secondary antibodies to initiate a reaction with the substrate. This reaction activates a light signal that can be detected through the exposure of the blot to x-ray film or through digital imaging by using a sensitive charged-couple device (CCD) imager.
The chemiluminescence method is the method of choice in most laboratories since it allows for the greatest sensitivity and produces faster results as compared to the other methods. It is also safe and convenient to use, can be used to strip and reprobe samples multiple times, and allows for easy documentation of results.
However, since the method relies on enzymatic reaction, the results can be affected by incubation and/or exposure time. In addition, it does not allow for the detection of more than one protein at a time since the reaction only produces one color light.
Fluorescence method
While the chemiluminescence method relies on the reaction of an enzyme (usually an HRP-conjugated enzyme) with the substrate, the fluorescence method uses a primary or secondary antibody labeled with a fluorophore dye to detect the protein of interest. In this method, a light source is used to excite the fluorophore. The resulting light signal is captured by a CCD camera to produce the final image and digitalized for data analysis.
The fluorescence method is highly sensitive, has a wide dynamic range and provides better linearity within detection limits. It produces quantitative results (resulting signal is proportional to the amount of protein present in the sample) and can be used in multiplex formats, even without stripping and reprobing. It also offers significant time savings as compared to the chemiluminescent method and produces less chemical wastes as compared to the other two methods.
Colorimetric method
With the colorimetric method, researchers usually use a secondary antibody that has been conjugated to an enzyme (horseradish peroxidase or alkaline phosphatase) to convert the dye into a colored precipitate or band that is readily visible on the membrane. The amount of dye converted into colored precipitate is directly proportional to the amount of protein present in the sample and is measured using a densitometer.
The colorimetric method is a simple and cost-effective technique that can be used to detect the presence of the protein of interest in a biological sample. It does not require a darkroom or any other expensive materials and the blots can easily be documented by photographing.
However, this technique is not as sensitive as the other two methods. In addition, it produces non-specific color precipitation, the blots fade upon exposure to light and the activity of the enzymes can be inhibited by the presence of azide.
