Western blot

Western Blot There are many “ Blot � techniques in molecular biology such as southern blot, northern blot, eastern blot and western blot. The principle and process of these blot techniques are similar. Southern and northern blot are nucleic acid blotting techniques, of which southern blot is for detecting a specific DNA sequence in DNA samples and northern blot is to study gene expression by detection of RNA (or isolated mRNA) in a sample. Eastern blot is used to analyze protein post translational modifications (PTM) such as lipids, phosphomoieties and glycoconjugates.

Different from those blot technique described above, western blot, also called protein immunoblot, is an analytical technique used to detect specific proteins in a given sample such as cell lysates, tissue homogenate or extract. It is widely used in molecular biology. The bases of western blot identification are two distinguishing properties: molecular weight and antibody binding specificity. It uses gel electrophoresis (usually SDS-PAGE) to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide.

And then transferred the protein sample to a membrane (typically nitrocellulose or PVDF), where they are probed with labeled-antibodies and stained with dyes for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection. Immunodetection involves the identification of a protein through its reaction with a specific antibody. Through spatial resolution, this method provides molecular weight information on individual proteins and distinguishes isoforms, alternate processing products, and other post-translationally modified forms.

The basic principle of western blot are protein electrophoresis and ELISA. Electrophoresis is a commonly used method for separating proteins on the basis of size, shape or charge. Proteins are large molecules with charge, they can migrate in the polyacrylamide gels under electric field. The migrating speed depends on the molecular weight of proteins: heavy proteins move slower than light proteins. http://www.creative-diagnostics.com/The-Basis-of-Western-Blot.htm