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Home >>Molecular Probes and Gene Libraries >> Southern, Northern and Western Blotting
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Southern, Northern and Western Blotting - A mixture of DNA, RNA or protein fragments can be separated by gel electrophoresis and the separated bands can be stained and visualized directly in the gel. However, to confirm the identity of these bands or to find similarity of one or more of these bands with a known and available molecular probe, it is possible to hybridize these bands with a labelled probe.

To facilitate this hybridization, the bands are often transferred to a nitrocellulose membrane through a technique described as blotting. When DNA bands are thus blotted, it is called Southern blotting (after the name of E.M. Southern); when RNA bands are thus transferred it is described by the jargon term Northern blotting and similarly when protein bands are transferred, the technique is described as Western blotting.

(a) Southern blotting. For Southern blotting, DNA sample is first digested with a restriction enzyme and digested sample is gel electrophoresed. The DNA bands in the gel are denatured into single strands with the help of an alkali solution. Subsequently, the gel is laid on top of a buffer saturated filter paper, placed on a solid support (e.g. glass plate), with its two edges immersed in the buffer.

A sheet of nitrocellulose membrane is placed on top of the gel and a stack of many papers (paper towels) on top of this membrane. A weight of about 0. 5 kg is placed on top of paper towels. The buffer solution is drawn up by filter paper wick, and passes through the gel to the nitrocellulose membrane and finally to the paper towels.

While passing through the gel, the buffer carries with it single stranded DNA, which binds on to the nitrocellulose membrane, when the buffer passes through it to the paper towels. After leaving this arrangement for a few hours or overnight, paper towels are removed and discarded.

The nitrocellulose membrane with single stranded DNA bands blotted on to it, is baked at 800C for 2-3 hours to fix the DNA permanently on the membrane. This membrane now has a replica of DNA bands from agrose gel, and can be used for hybridization with radioactively labelled DNA or RNA probe.

The membrane may then be washed to remove any unbound DNA and X-ray film is exposed to the hybridized membrane to get autoradiographs. The above steps involved in Southern blotting.

(b) Northern blotting. Initially the technique of Southern blotting used for DNA transfer from gel to the membrane, could not be used for blot-transfer of RNA. Instead mRNA bands from the gel were blot-transferred into a chemically reactive paper, prepared by diazotization of aminobenzyloxymethyl paper.

The technique being related to Southern blotting was called Northern blotting (not after name of any scientist as in Southern, blotting). Later, it was shown that mRNA bands can be blotted directly onto nitrocellulose membrane, a technique which has been widely adopted.

The mRNA bands blotted onto nitrocellulose membrane can be hybridized with a labelled DNA or RNA probe. The single stranded regions of the probe are removed by nuclease (mungbean nuclease or S-l nuclease), so that quantitative estimation of hybridized mRNA can also be made.

(c) Western blotting. This technique is used to detect proteins of a particular specificity. Particularly when a transferred gene expresses in transformed cells, the translated product in the form of protein can be identified by this technique. The extracted proteins are subjected to polyacrylamide gel electrophoresis (PGE) and are then transferred onto nitrocellulose, to which they bind.

Nitrocellulose membrane is then used for probing with a specific labelled antibody (antibody will not hybridize with protein, but bind with it). The antibody may be labelled with 1251 and the signal is detected again with autoradiography. If radioactive label is not used, bound antibody may be detected by a second antibody tagged with an enzyme.
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