GENE 500 Lecture Notes - Lecture 12: Chromogenic, Chemiluminescence, Electrophoresis
Document Summary
Immunoblotting, also called western blotting, combines the resolving power of gel electrophoresis with the specificity of antibodies. This multistep procedure is commonly used to separate proteins and then identify a specific protein of interest. Individual proteins (represented by blue ovals) are not visible at this stage. Step 2: the membrane is flooded with a solution of an antibody (ab1) specific for the protein of interest and allowed to incubate for a while. Then the membrane is washed to remove unbound ab1. Step 3: the membrane is incubated with a second antibody (ab2) that specifically recognizes and binds to the first (ab1). This second antibody is covalently linked to an enzyme that catalyzes a chromogenic reaction or releases light (e. g. , chemiluminescence), a radioactive isotope, or some other substance whose presence can be detected with great sensitivity. A sensitive method for tracking a protein or other biological molecule is by detecting the radioactivity emitted from a radiolabel introduced into the molecule.
