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Home >> Recombinant DNA Technology >> Western Blotting
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Western Blotting - In western blotting, proteins are electrophoresed in polyacrylamide gel, transferred onto a nitrocellulose or nylon membrane (to which they bind strongly), and the protein bands are detected by their specific interaction with antibodies, lectins or some other compounds. The various steps of this technique are briefly described below.
1. Protein bands are separated by polyacrylamide gel electrophoresis.
2. The protein bands are transferred onto a nitrocellulose or nylon membrane; initially this was achieved by a capillary movement of buffer similar to Southern blotting (capillary blotting), but nowadays it is usually done by electrophoresis (electrophoretic blotting).

Electrophoresis has been applied for the blotting step in Southern and northern hybridizations as well; in such 'cases buffer of low ionic strength (to avoid overheating during electrophoresis) and nylon membranes (since nucleic acids bind to nitrocellulose membrane only under conditions of high ionic strength) are used. The electrophoretic blotting, both of proteins and nucleic acids is much faster and more efficient than capillary blotting.
3. The specific protein bands are identified in a variety of ways.
(i) Antibodies are the most commonly used as probes for detecting specific antigens.
(ii) Lectins are used as probes for the identification of glycoproteins.

These probes may themselves be radioactive or a radioactive molecule may be tagged to them. Often the identification process is based on a 'sandwich' reaction.

In such an approach, a species specific second antibody or protein A of Staphylococcus aureus (protein A binds to certain subclasses of IgG antibodies) or streptavidin (it binds to biotinylated antibodies) is used to bind to the antibodies bound to the protein bands. These second molecules may be labelled with radioactive, enzyme or fluorescent tags; a single preparation of these labelled molecules can be employed as a general detector for various probes.

 
 

 
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