At TIPS journal club meeting held on January 21, 2019, the topic of “Western blotting technique: principle, procedure and application” was presented by Dr. Bayrami. A summary of this presentation is in below.
Western blotting technique is applied to detect the presence and alteration of a specific protein in a complex protein mixture and involves six steps. 1- Sample preparation: In this step the cells or tissue are washed, and lysed to release the target protein. 2- Gel Electrophoresis: The mixture of proteins migrate through a gel towards the positive pole at a rate proportional to their linear size and they are separated on the gel. 3- Blotting: Transferring the separated proteins on a membrane that can be Nitrocellulose or PVDF membrane. 4- Blocking: BSA is added to fill up the empty spaces of the membrane surface to prevent non-specific antibody binding to the membrane. 5- Antibody Probing: Primary antibody directly binds to the target protein. Then labeled secondary antibody binds to the constant region of the primary antibody. 6- Detection: the substrate solution is added to membrane and chemiluminescence reactions which are catalyzed by the attached label to antibody produces light or color that can be visualized and determined. Western blotting application in research includes: detecting changes in protein levels across time points, detecting tagged proteins, detecting truncated isoforms of proteins, detecting phosphorylation states of proteins and detecting the purity of drugs with protein structure.