Making of Antibodies

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Immunoprecipitation and Use of Antibodies in Isolation of Genes

One of the most useful discoveries of recent technology is the ability to isolate individual genes from an organism’s entire genome and then identify that gene. There are several methods available to achieve this goal, many of which make use of antibodies to identify potions of molecules. For proteins such as cell surface proteins, it is very difficult to purify a protein solution. To make antibodies to these types of proteins, whole cells or cell mixtures are injected into rabbits and the antibodies later collected. The antibodies must be separated from the other types in sera however. To accomplish this, techniques such as affinity chromatography are used to isolate antibodies form sera. Isolated antibodies can then be added to a protein solution, allowing the binding of particular proteins causing precipitation from solution. This type of isolation of target molecules from solution using antibodies is called immunoprecipitation. The proteins can then be removed from the antibodies and separated using gel electrophoresis techniques. A very common technique often used is called two-dimensional gel electrophoresis. A sample is run on a very thin strip of polyacrylamide then placed under a perpendicular current, moving the proteins within the sample first in one direction, then separating them in another. This allows separation of molecules by size and by differing charge of molecules of similar molecular weights.

Most useful to the fields of biochemistry and molecular genetics is the use of these methods in gene identification. First a gene must be isolated from an organism. This can be accomplished using restriction enzymes, cutting the DNA into pieces and then inserting these pieces into plasmid vectors, creating a library of genes. These vectors are then inserted into bacteria, which proceed in replicating the genes and producing their products. Any bacteria producing the protein of interest are isolated, using radiolabeled antibodies which bind specifically to the target protein. Transfected bacterial colonies are washed in these labeled antibodies. The remaining antibodies are then washed off as those having a complementary protein are kept on the surface. The colonies are then observed for radiolabeling. Any colony exhibiting radioactivity has a protein product able to bind to the specific antibody. These colonies can then be removed and isolated. Their inserted genes can then be removed and sequenced, giving the genetic code for the DNA responsible for a particular protein of interest.
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