Immunoblotting, also known as Western blotting, is a laboratory technique used to detect specific proteins in a given sample. The technique is based on the transfer of proteins separated by electrophoresis (a technique used to separate proteins based on their size) onto a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. The proteins are then probed using specific antibodies to detect the presence and quantity of a target protein. The technique is commonly used in molecular biology, biochemistry, and clinical analysis.
The immuno-blotting technique involves the following steps:
1. Sample preparation: The sample to be analyzed is first prepared by solubilizing the proteins using a lysis buffer.
2. Protein separation: The proteins are separated based on their size using electrophoresis. The most commonly used electrophoresis technique is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins according to their molecular weight.
3. Transfer of proteins: The separated proteins are transferred from the gel onto a nitrocellulose or PVDF membrane using electrophoresis or semi-dry transfer. The membrane is then blocked with a blocking solution to prevent non-specific binding of antibodies.
4. Primary antibody incubation: The membrane is incubated with a primary antibody that specifically recognizes the target protein.
5. Secondary antibody incubation: The membrane is then incubated with a secondary antibody that recognizes and binds to the primary antibody.
6. Visualization: The target protein is detected using detection methods such as chemiluminescence or fluorescence.
The immuno-blotting technique has several advantages over other protein detection techniques. It is highly specific, sensitive, and can detect proteins present in low concentrations. It can also be used to identify different isoforms and post-translational modifications of a protein. However, it requires careful optimization of the antibody concentrations and the blocking conditions to achieve optimal results.