Biology 2382B Study Guide - Hspa8, Calnexin, Calreticulin
3 Mar 2014
School
Department
Course
Professor
Document Summary
Western blotting (or immunoblotting) is a process used to detect specific proteins. First, an electric current is used to transfer the proteins on the sds-polyacrylamide gel onto a membrane. This membrane is then incubated with primary antibodies that recognize your protein of interest. Enzyme-labelled secondary antibodies are then linked to the bound primary antibodies. Finally, a substrate is added to the membrane that is catalyzed by the enzyme and gives off luminescence (enhanced chemiluminescence), showing you where your protein of interest is located. This last step is very rarely used and more often fluorophores are attached to the secondary antibody. The luminescence makes an impression on the x-ray film and you can see darker banding patterns indicating higher expression. In a typical mammalian cell, tens of thousands of different proteins are made and it becomes a major issue for the cell to direct them to the right destination.
