Western Blot Western Blot Western blotting is to protein, as Southern blot is to DNA, and RNA is to Northern blotting. In all blotting techniques, samples are separated electrophoretically and are transferred from a gel to a membrane. The membrane is then probed with factors which are specific for amino acids (western) or nucleotides (Southern and Northern)
Antibodies for Western Blot Western blot uses antibodies which act as probes. The antibodies react specifically with antigenic epitopes on the target protein which are attached to the solid support. Western blotting is an important technique for the detection and quantification of proteins that are not radiolabeled. Western blotting can detect as little as 1 ng of protein an average sized protein. The major difference between Western blotting and Northern / Southern blotting is the properties of the probes used in the detection of proteins. Whereas nucleic acids are used as probes in the other blotting techniques, western blotting uses antibodies to probe for target proteins of interest. Antibodies behave much differently however than nucleic acid probes. Western blotting or Immunoblotting is used to determine the size and quantity of protein antigens which react with a specific antibody. Electrophoresis first separates the proteins through an SDS-polycacrylamide gel and then the proteins are transferred electrophoretically from gel to membrane.
Types of Western Blotting Membranes: - Nitrocellulose
- Nylon and positively charged membranes
- PVDF polyvinylidene fluoride
Also, PVDF must be soaked in 95% methanol (also sometimes isopropanol or ethanol) prior to usage. PVDF membranes also tend to be thicker and more resistant to damage during use. |
