Blotting Techniques

What happens then is that the protein is expressed by the animal or bacterial cell. It is expressed in high levels because multiple copies of the gene are inserted so that a great deal of the desired protein can be extracted from the bacterial cell or animal cell. It can be tested for enzymatic activity if it is an enzymatic protein and can be crystallized to make a drug that could be useful to the pharmaceutical companies. Theoretically, patients with an enzyme deficiency could be inoculated with a plasmid containing the missing enzyme and could be cured because they now have the DNA necessary to make the missing enzyme. Another commonly-used technique in molecular cell biology is the polymerase chain reaction or PCR. This is a way of copying sequences of DNA. The technique is so successful that more than a billion DNA molecules can be isolated from a single strand of DNA in under two hours. The technique can be used to create mutations on existing DNA or to add sections of DNA coding for specific enzymes on the ends of normal chromosomes. Mutating DNA by this method is called “site-directed mutagenesis”. There are different PCR techniques that can identify amplify both DNA and RNA, making protein synthesis possible. Molecular cell biologists also make use of gel electrophoresis. The idea is the different lengths of DNA, proteins, and RNA migrate differently along an electrical field. Gel is used and strands of DNA are placed on one end of the gel. An electrical field is applied to the gel and the strands migrate a certain distance, depending on their size and electrical charge. These can be stained and identified by laboratory scientists as ways to find certain strands of DNA, RNA, or proteins.

There are four different blotting techniques used by molecular and cell biologists. The first is the Southern blot. It is a technique that probes for a certain DNA strand on a sample of DNA. The DNA is sometimes digested by restriction enzymes (which is also called restriction endonuclease digestion) and is then separated using gel electrophoresis. The DNA is taken up by a membrane by means of capillary action before being exposed to a labeled probe that matches the DNA segment of interest. If the DNA segment of interest is present, it will stain appropriately. This technique is less effective than PCR so it isn’t used as often as it used to be. The Northern blot technique is used to understand expression patterns of different types of RNA molecules compared to a standard set of RNA molecules. This combines gel electrophoresis of RNA and a blotting technique. RNA is separated by the gel procedure and placed on a membrane, where it is labeled to identify a sequence of interest. It is a test that measures gene expression because genes that are being expressed will make RNA, while genes that aren’t being expressed won’t make RNA. Western blotting techniques involve the separation of proteins by gel electrophoresis by size and electrical charge. The proteins are then placed between two glass plates and transferred to membranes that can be probed with antibodies that are stained or otherwise tagged. This procedure sometimes uses chemiluminescence and enzymes to allow for the qualitative and quantitative evaluation of certain proteins in tissue or cell sections. Eastern blotting also measures proteins but it measures the post-translational modification of proteins. Substrates are used to probe for proteins that have been blotted onto membranes