Re: Northern and Western blotting

The terms "nothern" and "western" blots derive from the "Southern" blot (note the capitalization) as invented by E. M. Southern in the 1970s.

In a Southern blot, fragments of the DNA are first separated by size using gel electrophoresis. The DNA has generally been digested with a restriction endonuclease to create fragments of defined sizes. Restriction endonucleases only cut DNA at specific sites, thus their ability to create DNA fragments of defined length. A sample of digested DNA is then placed at one end of a "gel" made of agarose (~clear jello). When an electric field is applied, the negatively charged DNA migrates through the gel towards the positive pole of the electrophoresis rig. Smaller segments DNA have an easier time moving through the agarose matrix, and so the smallest fragments of the DNA move the furthest from the point at which they were loaded onto the gel. In this manner molecules of DNA can be separated by size.

After electrophoresis, the gel is removed from the rig, and the DNA transferred onto a membrane. Nitrocellulose membranes were originally used, but they behave rather explosively when exposed to open flames; newer nylon mesh membranes are now used.

The membrane, with associated pieces of DNA, can now be *probed* with a specific DNA sequence. Since the electrophoresed DNA has been fractionated by size, if the probe recognizes a single unique sequence of DNA it will bind to a specific site on the membrane. Probes are usually labeled in some manner, often with radioactive isotopes (32P or 35S, e.g.). The probed membrane can then be placed under film and developed. Dark bands will appear where the radio-labeled probe bound sequnces of DNA stuck to the membrane.

"Northern" and "western" blots follow the same procedure as the Southern blot - size fractionate, transfer to a membrane, then probe - but use different starting materials. The names were used in directional "honor" of the "Southern" blot (who says science can't poke fun at itself?). In a northern blot molecules of RNA are size fractionated then probed with a complementary DNA or RNA probe. In a western, proteins are size fractionated on a gel commonly made of acrylamide, transferred to a membrane, then probed with antibodies that should recognize some determinant on the protein.