Blotting

Blotting is a common laboratory procedure in which biological molecules in a gel matrix are transferred onto nitrocellulose paper for further scientific analysis. The biological molecules transferred in this process are DNA fragments, RNA fragments, or proteins. Because the isolation and characterization of these types of materials is at the center of much molecular biology research, blotting is one of the most useful techniques in the molecular biology laboratory.

The blotting procedure is named differently depending on the type of the molecules being transferred. When the molecules to be transferred are DNA fragments, the procedure is called a Southern blot , named for the man who first developed it, Edward Southern, a molecular biologist at Oxford University. The Northern blotting procedure, which transfers RNA molecules, was developed shortly thereafter and, since it was patterned after Southern blotting, its name was a humorous play on words inspired by the name of the first procedure. Western blotting got its name in a similar fashion. All three blotting methods are relatively easy to carry out, can be conducted in a short period of time, and provide answers to many questions that are commonly raised in the field of molecular biology.

The Procedure

All blotting procedures begin with a standard process called gel electrophoresis . During this step, DNA, RNA, or proteins are loaded on to an agarose or acrylamide gel (that functions like a molecular sieve) and are then run through an electric field. Two types of gels are commonly used: agarose gels and acrylamide gels. Agarose gels are based on a meshwork of agar filaments and are most often used to analyze DNA and RNA. Acrylamide gels are based on a meshwork formed from the chemical acrylamide and used most often to analyze proteins. Gels are loaded with a mixture of many differently sized molecules. When pulled through the gel by the electric current, they will separate into separate pools on the basis of their size; smaller molecules migrate farther through the gel than larger molecules. These separate pools of molecules will appear as bands on the gel if they are stained with an appropriate dye. After the molecules have been fractionated on the gel, they are ready for transfer to the nitrocellulose paper.

Transfer is initiated when the gel is retrieved from the electrophoresis apparatus and the nitrocellulose paper is carefully laid on top of the gel. The objective now is to transfer the bands of molecules found in the gel over to the nitrocellulose paper. Here they become immobilized, and will reflect the pattern seen on the gel. The paper now serves as a type of permanent record of the gel's banding pattern that can be used for further analysis.

There are two basic ways the actual transfer, or blotting, is carried out. One method takes a "sandwich" of gel and nitrocellulose paper and places it in a special apparatus that sets up an electric field running perpendicular to the band as preserved in the gel. This pulls the bands of molecules out of the gel, and they are immediately absorbed onto the nitrocellulose paper. This method is most commonly employed in Western (protein) blots.

The other method, commonly employed with Southern and Northern blots, lays the gel on top of a platform that in turn is placed in a tray containing a buffer solution. Underneath the gel is a strip of blotting paper that is folded down on each side of the platform, so that it dips into the buffer to serve as a wick. On top of the gel are placed, first, the strip of nitrocellulose paper, then several pieces of blotting paper, and finally a small stack of paper towels. A weight is then placed on top of the paper towels. The buffer flows up the blotting paper "wick" by capillary action, then through the gel, through the nitrocellulose paper, and ultimately into the paper towels. The DNA or RNA in the gel moves with the buffer but sticks to the nitrocellulose paper on contact. The paper towels soak up the transfer buffer, but only after it has passed through the gel and deposited the DNA or RNA on the nitrocellulose paper.

After transfer has been completed, the nitrocellulose paper can be examined by using probes. Short fragments of DNA that have a nucleotide sequence complementary to the molecule being analyzed are normally used as probes in Southern and Northern blots. Antibodies that react with the protein being analyzed are used as probes in a Western blot. In either case, the probe is "labeled," usually by making it radioactive, so that it is easy to identify. In all blotting experiments, the nitrocellulose paper is placed in a chamber full of buffer and mixed with the probe, which then binds to the molecule that is being studied. This is called the hybridization step. Detection of the probe indirectly detects the molecules being studied.

Illustrative Examples

Blotting is perhaps best understood with illustrative examples. Suppose a student was studying a newly identified gene, X, from cows. The student then asks three basic questions as part of a research project: (1) Do pigs also have gene X on their chromosomes? (2) Do cows express gene X in their brain tissue? (3) Is the protein product of gene X found in the cow's blood plasma? Blotting experiments can answer all three of these questions.

A Southern (DNA) blot will answer the first question. The student obtains DNA from a pig, uses a restriction enzyme to cut the DNA into a large pool of fragments of different sizes, and then fractionates the DNA fragments using gel electrophoresis. The contents of the gel are then chemically treated so that the double-stranded DNA molecule "unzips" and exists in a single-stranded form, which is then blotted onto nitrocellulose paper. At this point, the student can take gene X (or a portion of the gene) from the cow, label it, make it single-stranded, and use it as a probe to analyze the pig's DNA. The labeled probe is then added to the nitrocellulose blotted with the pig DNA. If the pig's DNA also contains gene X, there should be a fragment on the nitrocellulose with a nucleotide sequence sufficiently complementary to the probe such that the probe will bind. In other words, the labeled probe will bind to any fragment from the blotted pig DNA that contains gene X, allowing the student to detect the presence of gene X in pigs.

To answer the second question, a Northern (RNA) blot would be used. The procedure is essentially the same as with the Southern blot, except that the student would isolate RNA from the cow's brain tissue and run it out on the gel. The same DNA probe described above would then be used to detect whether the RNA that represents gene X expression is present in the brain.

To answer the third question, the student would use a Western (protein) blot. This requires the use of an antibody that specifically reacts with the protein coded for by gene X. The student first obtains plasma from the cow and uses standard biochemical techniques to isolate the proteins for analysis. These proteins can then be run out on a gel and transferred to nitrocellulose. The proteins can then be probed with the labeled antibody. If the product of gene X is in the plasma, it will bind with the labeled antibody and can thus be detected.

see also Gel Electrophoresis; In situ Hybridization; Restriction Enzymes; Sequencing DNA.

Michael J. Bumbulis

Bibliography

Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.

Russell, Peter. Genetics, 5th ed. Menlo Park, CA: Benjamin Cummings, 1998.

Watson, James D., et al. Recombinant DNA, 2nd ed. New York: Scientific American Books, 1992.