Western Blotting

Western Blotting, also known as immunoblotting, is a widely used method/technique to detect and/or quantify specific proteins from the sample of interest. The procedure involves a few stages. The protein sample is initially loaded and run on polyacrylamide gel electrophoresis wherein a voltage is applied and the protein separates based on their charge/mass ratio. A reference protein ladder is used to assess the molecular weight of proteins in the sample. The proteins are then transferred from the gel onto a membrane usually made of nitrocellulose or polyvinylidene difluoride (PVDF). This transfer is accomplished by electric current in electroblotting or by capillary action through the filter papers. A primary antibody binds to the protein of interest (now on the membrane). In some procedures, a secondary antibody is also used which binds to the primary antibody's Fc region. Various methods can be used for final detection of antibody bound protein of interest like Chemiluminescent detection or Fluorescent detection depending on the probe.

 

Western Blotting is a highly sensitive method for protein detection. In diagnostics, it is employed in detecting anti-HIV antibody and Hepatitis B infection. In molecular biology, it can detect the presence of phosphorylated proteins, tagged proteins or various isoforms of proteins. In genetics, it can be used for gene expression studies amongst others.