Blotting analysis

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Blotting analysis

Blotting analysis describes a series of techniques used to determine and describe protein and nucleic acid. Blotting techniques are applicable to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

Blotting analysis allows scientists to transfer electrophoretically separated components from a gel, which is used to separate the different proteins or enyzmatically-generated fragments of nucleic acid, to a solid support (nowadays, typically a nylon membrane). This support may then be used for probing with reagents specific for particular sequences of amino acids or nucleotides. In this way, the size and/or quantity of the proteins or nucleic acids under study can be gauged.

Blotting analysis was originally developed in 1975 by British molecular biologist E. M. Southern while he was on a leave of absence from his Edinburgh lab to do research in Zurich. This process has since been referred to as Southern blotting. Southern’s method was designed to transfer fragments of DNA from the agarose gel in which they had been separated onto cellulose nitrate filters. The DNA fragments are deposited onto a filter laid over the gel as a result of capillary action, which is established and maintained by the flow of buffer from underneath the gel to a stack of dry paper towels placed on the filter.

Additional methods have been developed for the transfer of DNA. Electrophoretic transfer can be performed by mounting the gel and membrane between porous pads aligned between parallel electrodes in a tank containing buffer of high ionic strength. The electric current drives the transfer. However, resulting high temperatures require that the tank be cooled. This method is most often used with gels made from polyacrylamide, not agarose, since polyacrylamide has a higher melting temperature. A third method involves the use of a vacuum. The gel is placed in contact with the membrane on a porous screen above a vacuum chamber, and a buffer elutes the DNA from the gel onto the membrane.

After the DNA is deposited onto the solid support, the filter is usually dried at a high temperature in order to bind the nucleic acid strongly to the membrane. Alternatively, the DNA can be covalently attached to the filter by cross linking with low doses of ultraviolet radiation. The process of attaching the nucleic acids allows the filter to be sequentially hybridized to several different probes with little loss in sensitivity.

Originally, the most common solid supports used were nitrocellulose filters. However, the filters become brittle when dried, and as a result, they can not be used for more than one or two cycles of hybridization. Currently, charged nylon membranes are a popular choice, as the DNA binds irreversibly and the membrane is more durable to withstand multiple hybridizations.

Once the DNA is bound to the membrane, the process of hybridization may begin. Typically, the membrane is first incubated with a buffer designed to limit the amount of non-specific binding of the chosen probe to parts of the membrane not covered by DNA. Next, a radiolabeled probe is added. This probe is complementary to sequences of the DNA of interest and therefore binds those sequences with a given affinity. The sort of probe used can vary greatly, from purified RNA to cloned complimentary DNA (cDNA; a DNA copy of a target stretch of RNA) to short synthetic oligonucleo-tides. Unbound probe is then washed away from the membrane. Autoradiography of the labeled blot results in visualization of bands that correspond to the probe sequence. The size of the bands can be determined by their placement along the length of the gel in comparison with markers of known size. Additionally, the strength of the band seen can also be used to quantify the amount of DNA present.

Southern blotting has many uses in the field of molecular biology. Genomic Southern blots can provide a physical map of restriction sites within a gene in a chromosome through the analysis of fragments produced after digestion of genomic DNA with one or more restriction enzymes. Other uses include detecting major gene rearrangements and deletions involved in disease, identifying structurally related genes within the same species or homologous genes in other species, and more recently, screening genomes of various mutagenized lines in order to identify a mutant in a particular gene under investigation.

After the development of Southern blotting, similar procedures were created to analyze RNA as well as proteins. Named Northern and Western blotting, respectively, these techniques have many similarities to Southern blotting but require several modifications.

Northern blotting refers to the transfer of RNA to a solid support from a denaturing agarose gel. The membranes and transfer apparatus used are similar to those used in Southern blotting. The RNA run in the gel may be total RNA isolated from particular samples. Alternatively, RNA containing poly(A) tails, a characteristic of messenger RNA (mRNA), which is involved in the transfer of genetic information from DNA to protein, may be purified from the total RNA sample in order to analyze specifically mRNA molecules. As with Southern blotting, radiolabeled RNA, DNA, or oligonucleotides are used as probes, and the blots are hybridized and autoradiographed in a similar manner. More recently, additional methods of detection that do not require radioactivity are commonly used for both northern and Southern blotting. Northerns can be used to determine the size or amount of specific mRNAs, to examine in what tissues or under what conditions certain genes are expressed (i.e., are copied in the form of mRNA), and to study conditions that alter the level of particular mRNAs.

The analysis of proteins is accomplished by western blotting. Generally, proteins are put in solution (solubilized) with detergents and reducing agents, separated

KEY TERMS

Northern analysis— A technique used to identify and locate mRNA sequences that are complementary to a piece of DNA called a probe. Molecular biologists commonly refer to this as a “Northern blot.”

Southern blot— Transfer by absorption of DNA fragments separated in electrophoretic gels to membrane filters for detection of specific base sequences by radiolabeled complementary probes.

Western blot— A technique used to identify a specific protein; the probe is a radioactively labeled antibody that binds the protein in question.

in polyacrylamide gels, and transferred to a nitrocellulose filter or polyvinylidene fluoride membrane, where they become covalently bound to the support. The crucial difference between westerns and Southerns is the probe. For western blots, specific unlabeled antibodies, which typically must be produced for each individual protein and which react specifically with their target, are used to recognize the protein of interest from within the background of other cellular proteins. Antibodies, unlike their nucleic acid probe counterparts, do not bind with predictable rates or specificities, and therefore require increased levels of optimization in order to extract the most useful information. The bound antibody is detected on the blot by one of several secondary reagents that is either radiolabeled or coupled to an enzyme. Activity of the enzyme in the presence of its substrate allows the detection of antibodies bound to the protein of interest. Westerns can be used to search for the presence of certain proteins in specific samples, tissues, or treatments. They may also be used for quantitative analyses or to determine the apparent molecular weight of a protein.