Question:
How to detect protein using western blotting?
The Protein Man Says:
So, how does the fluorescence and colorimetric method of protein detection differ from the chemiluminescence method? Here are some things you may need to know.
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.
