Western Blot

The western blot test, also known as protein immunoblot test, is a technique, whereby specific protein samples can be detected from specimens of tissues. It helps in determining the molecular weight of the protein and the quantity of protein present in different samples of tissues. It detects and defines the nature and characteristics of proteins, by studying their ability to bind specific antibodies.

History of western Blot test:

This technique was invented in the laboratory of George Park in Stanford. W. Neal. Burnette named this technique as Western Blot.

Steps involved in Western Blot test:

1. The first step involves, separating the protein with the help of a gel electrophoresis.
2. Then the proteins are collected in nitrocellulose blotting papers. Other types of materials or membranes may also be used. However, it has to be kept in mind that the proteins retain its property and the pattern of separation.
3. The blot is then put under incubation with milk protein or any other generic protein.
4. An antibody is poured in the mixture, so that it binds to the specific protein in that solution.
5. The location of the antibody is determined by putting it under incubation with a colorless substrate.
6. Then the enzyme in the antibody turns colored.

Uses of Western Blot:

• It detects special protein of interest from a number of other proteins, in that sample of tissue
• It can determine the size of the protein
• It gives idea about protein expression.
• It can study protein samples from both cells and tissues.
• This is a very typical test in the fields of microbiology, genetics, immuno-genetics, biochemistry and bio- physics.

The western blot test depends a lot on the quality of antibody that is used, and how compatible is it for the protein in the sample of tissue or cells. This would facilitate the binding of the protein with the antibodies.

Where can you get the materials for Western blot test?

• The nitrocellulose blotting paper is available in any laboratory and in most medical stores.
• The antibodies are also available in shops.
• If commercial antibodies are expensive, then one can resort to reutilization of unbound antibodies.

There are three types of Western blot test:

1. Dot Blot test:
2. 2D Blot Test:
3. ELISA test

In case of HIV, one generally relies on the ELISA test. In this Western Blot Test, blood is taken from vein in the patient’s arm. Then the area is cleaned with antiseptic. The administration of the needle is not at all painful in most cases. This blood sample is then tested. ELISA is very useful for the detection of certain infections and diseases like Kaposi’s Sarcoma and pneumocytis pneumonia. It detects the possibility of HIV virus in the protein molecules of the patient’s blood cells.

However, a negative Western Blot test does not mean that the person is HIV free. The patient may be in the first phases of the disease, when not too many HIV antibodies are produced to harm the blood cells. It is also not wise to believe that every positive Western Blot test means HIV. It may be the presence of curable diseases like Lyme disease or syphilis.

Therefore, just one Western Blot test should not be done. A few regular one, conducted in a span of two weeks, can yield the correct result.