What is the difference between a Southern blot, a Northern blot, and a Western blot?
Include a basic overview of the procedures.
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Southern blotting was the first type of blot developed, and it was named after its inventor, E.M. Southern.
Southern blotting involves DNA.
Northern blotting involves RNA.
Western blotting involves protein.
All three techniques involve isolating the macromolecules of interest (RNA, DNA or protein) away from the rest of the cellular constituents. Genomic (and mitochondrial) DNA must be cut into smaller pieces using restriction enzymes. All three procedures require denaturation of the macromolecules by heating. The samples then undergo gel electrophoresis to separate based on size. The largest molecules stay near the top of the gel, the smallest are able to travel further through the porous gel.
Next, the macromolecules are transferred from the gel onto a nitrocellulose membrane.
The membrane is probed using: a radio labeled complementary DNA sequence for Southern and Northern, or an antibody recognizing the protein of interest (primary antibody) for Western blots. Western blots require an addition of a secondary antibody that recognizes the primary and is radio labeled.
Finally, the membrane is exposed to film, the film is developed and a band appears wherever the molecule of interest was on the membrane. (See the attachment below)
The attached file shows a Western I ran looking for the protein MUC1. MUC1 is a mucin protein, so it is very large and it runs near the top of the gel.
(Note: other methods that do not require radioactivity have been developed, where an enzyme is attached to the probe, rather than a radioisotope. When the proper substrate for that enzyme is added, the product gives off a signal that can be detected by exposing and developing a film)