Posted on April 21, 2021
Antibody Classes or Isotypes
Western blots are the SDS-PAGE gel that goes one step further. SDS-PAGE is a protein separation process based on size. The protein is denatured and loaded onto the polyacrylamide gel, and an electric field is applied which causes the smaller protein to move faster than the larger protein through the gel to the positive electrode. You can get western blotting service from various sites over the internet.
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After sufficient protein has been departed on the gel, they can be transferred from the gel to the nitrocellulose or PVDF membrane. This is the first step in performing a western blot. The layer can be blocked with BSA or milk and then exposed to primary antibodies that bind to certain proteins on the membrane.
The secondary antibody binds to the Fc region of the primary antibody. Secondary antibodies are conjugated to enzymes that can catalyze chemiluminescent reactions such as horseradish peroxidase.
When a substrate is added to the membrane, the desired protein can be visualized as a separate band on the membrane. The membrane is usually exposed to the film and the lines are immortalized as dark lines on the film.
Since the primary antibody is specific for the desired protein, Western blots can be used as an identity test. The molecular weight of the protein confirms its identity. PBL can implement the Western Blot as a cGMP process, as part of a GLP survey or as an R&D project.