What are the steps in Western blotting?

Western blotting is a protein detection technique first invented by American George Stark. It uses electrophoresis technology to separate proteins from specific tissues or cells, fix them on NC membranes or PVDF membranes, and then uses specific antibodies to detect specific antigens. Because this technology can find antigens that cause diseases, it has now been widely used in many biological fields such as disease diagnosis. Let’s take a look at the steps for making pig’s trotters.

1. Principle

It is similar to Southern Blot or Northern Blot hybridization methods, but Western Blot uses polyacrylamide gel electrophoresis, the object to be detected is protein, the "probe" is antibody, and the "development" is a labeled secondary antibody. The protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier (such as nitrocellulose membrane). The solid phase carrier adsorbs the protein in the form of non-covalent bonds and can keep the type of polypeptide separated by electrophoresis and its biological activity unchanged. The protein or polypeptide on the solid phase carrier is used as the antigen, which reacts with the corresponding antibody, and then reacts with the second antibody labeled with an enzyme or isotope, and is detected by substrate color development or radioautography.

2. Classification

There are several main methods for Western Blot development:

i. Autoradiography

ii. Substrate chemiluminescence ECL

iii. Substrate fluorescence ECF

iv. DAB substrate color development

The commonly used methods are substrate chemiluminescence ECL and substrate DAB colorimetry. The first method is used for the same level and experimental conditions, and published articles usually use substrate chemiluminescence ECL. All you need to do is buy a ready-made test kit, and the operation is relatively simple. The principle is as follows (the secondary antibody is labeled with HRP): the reaction substrate is peroxide + luminol. When it encounters HRP, it will emit light, which can expose the film and wash out the bands. Measure the protein components expressed by specific target genes separated by electrophoresis. This technique is also widely used to detect protein levels.

3. Sample preparation

1. Extraction of total protein from monolayer adherent cells:

2. Extraction of total protein from tissues:

3. Extraction of total protein from drug-treated adherent cells.

Western blotting, a method for identifying diseases caused by misexpressed genes. This advantage of being able to quickly find the antigen that causes the disease and having the specific antibody (a protein) stained is widely used in the study of genetic diseases, genetic problems, and disease detection. Based on its production, new detection technologies are also being developed, such as the Southern Blot hybridization method.