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Different Techniques of BlottingBY: Amna Adnan | Category: Biotechnology-products | Submitted: 2010-10-20 02:00:19
Blotting is a technique for detecting DNA, RNA or proteins initially present in a complex mixture. In this technique the molecules separated by gel electrophoresis are transferred to nitrocellulose filter. Three types of blotting techniques are commonly used for visualizing particular macromolecules:
The procedure of Southern blotting was first developed by Edward Southern and was named after his name. Southern blotting is used to detect DNA from a complex mixture.
First the DNA is extracted from the cell. The extracted DNA is than cut up with restriction enzymes. The resulting DNA fragments are separated by gel electrophoresis, in which the molecules spread out from one end of the gel to the other. For blotting technique the agarose gel is first washed with buffer solution to remove the accumulated contaminant or agarose residues. Gel is carefully removed from the glass plate and is placed in the transfer buffer. In the mean time nitrocellulose membrane and blotting papers are cut according to size of gel. Finally nitrocellulose membrane is placed on the gel above which blotting papers dipped in transfer buffer are placed. Five to six pieces of blotting papers are placed on each side. Gel with nitrocellulose membrane and blotting papers is then rolled with glass rod to remove any trapped air. Now stacks of unsoaked blotting papers are placed above it. Finally glass plate is placed above them. This set up is transferred to reservoir containing transfer buffer. The transfer buffer from the reservoir moves to the blotting paper by capillary action. The set up is allowed to stand for 12 to 13 hours. In this time the DNA from the gel is transferred to the nitrocellulose membrane. This immobilization is because of the negative charge of DNA and positive charge of nitrocellulose membrane. As soon as the transfer is finished, nitrocellulose membrane is carefully taken out from the setup. Now the membrane will have DNA fragments in the same pattern as they were on the gel. All the bands present in the membrane are marked with pen or pencil. Membrane is than exposed to ultraviolet rays or at 80⁰C in a vacuum oven. This is done to strengthen the binding of DNA fragments to nitrocellulose membrane. This will further not allow DNA to wash off while membrane washing. The membrane can now be incubated with the specific probe of interest. Once the incubation is complete, it can be detected by autoradiography.
The procedure of Northern blotting is exactly similar to Southern blotting. The only major difference between the two is that in Northern blotting RNA is transferred onto the membrane from the gel.
The procedure of western blotting is also similar to Southern blotting. But in western blotting proteins are separated instead of DNA. In Western Blotting there is no need to expose nitrocellulose membrane to ultraviolet ray or vacuum. Instead antibodies are used to detect the presence of proteins. Primary antibody binds to paper while secondary antibody binds to the primary antibody. Blot is incubated with the primary antibody of interest. Non bound antibodies are usually washed off from the membrane. The presence of bound antibody is detected by incubating it with secondary antibody of interest. Secondary antibody is usually attached with an enzyme so that its presence can be detected on a nitrocellulose membrane. Secondary antibody can also be attached with radioactive isotopes. The color emitted determines the presence of particular protein.
Blotting techniques have wide application in today's world. Southern Blotting helps in determining the nucleic acid sequence of animal or human being while Northern blotting is helpful in gene expression.
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