ImmunologyMolecular Biology

Western Blotting

Western Blot

An immunoblotting technique which rely on the specificity of binding between a protein of interest and a probe (antibody raised against that particular protein) to allow detection of the protein of interest in a mixture of many other similar molecules is western blotting.

Western blot is the analytical technique used in molecular biology, immunogenetics and other molecular biology to detect specific proteins in a sample of tissue homogenate or extract. It is used for the detection of proteins and also called protein immunoblotting because an antibody is used to specifically detect its antigen.

Principle

The technique consists of three major processes:

  1. Separation of proteins by size (Electrophoresis).
  2. Transfer to a solid support (Blotting)
  3. Marking target protein using a proper primary and secondary antibody to visualize (Detection).

 

Proteins are electrophoresed in polyacrylamide gel, transferred onto a nitrocellulose or nylon membrane and the protein bands are detected by their specific interaction with antibodies, lectins or some other compounds.

 

The antibody can be labelled with I125 and the signal is detected against by using autoradiography. If radioactive label is not used, the bound antibody may also be detected by a second antibody tagged with an enzyme (as in ELISA).

 

Electrophoresis used to separate proteins according to their electrophoretic mobility which depends on charge, size of protein molecule and structure of the proteins. Proteins are moved within the gel onto a membrane made of Nitrocellulose (NC) or Polyvinylidene difluoride (PVDF). Without pre-activation, proteins combine with nitrocellulose membrane based on hydrophobic interaction (Blotting). For detection of the proteins primary antibody and enzyme conjugated secondary antibody are used. On addition of substrate, a substrate reacts with the enzyme that is bound to the secondary antibody to generate colored substance, namely, visible protein bands.

 

Western Blot

 

Western blotting is usually done on a tissue homogenate or extract. It utilizes SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis), a type of gel electrophoresis to first separate various proteins in a mixture on the basis of their shape and size. The protein bands thus obtained are transferred onto a nitrocellulose or nylon membrane where they are probed with antibodies specific to the protein to be detected. The antigen–antibody complexes that form on the band containing the protein recognized by the antibody can be visualized in a variety of ways.

 

If the protein of interest is bound by a radioactive antibody, its position on the blot can be determined by exposing the membrane to a sheet of X-ray film, a procedure called autoradiography.

 

However, the most generally used detection procedures employ enzyme-linked antibodies against the protein. After binding of the enzyme–antibody conjugate, addition of a chromogenic substrate that produces a highly colored and insoluble product causes the appearance of a colored band at the site of the target antigen. The site of the protein of interest can be determined with much higher sensitivity if a chemiluminescent compound along with suitable enhancing agents is used to produce light at the antigen site.

 

Applications:

  1. Identification of a specific protein in a complex mixture of proteins. It show the presence of specific protein both by size and through binding of an antibody, which makes them well suited for evaluating levels of protein expression in cells, and for monitoring fractions during protein purification.

 

  1. It is most widely used as a confirmatory test for diagnosis of HIV, where this procedure is used to determine whether the patient has antibodies that react with one or more viral proteins or not. Also used as the definitive test for Bovine Spongioform Encephalopathy (BSE)

 

  1. Demonstration of specific antibodies in the serum for diagnosis of neurocysticercosis and tubercular meningitis.

 

  1. Estimation of the size of the protein as well as the amount of protein present in the mixture.

 

  1. To compare expression of a target protein from various tissues.

 

  1. Quantifying a gene product (gene expression studies).