Using Western Blotting for Protein Detection: Some Commonly Used Methods (Part 1)

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.

We will be discussing two more methods of protein detection in our next post so please watch out for it.