WESTERN BLOT
Western blot analysis is used to detect a protein in a mixture of any number of proteins while giving you information about the size of the protein. This method is, however, dependent on the use of a high-quality antibody directed against the desired protein. The antibody is used as a probe to detect the protein of interest.
Western blot allows investigators to determine the molecular weight of a protein and to measure relative amounts of the protein present in different samples. This is an analytical method that involves the immobilization of proteins on membranes before detection using monoclonal or polyclonal antibodies. In western blotting which is also called immunoblotting, prior to protein immobilization on the nitrocellulose membranes, sample proteins are separated using SDS polyacrylamide gel electrophoresis, providing information about molecular weight and the potential existence of different isoforms of the proteins under study.
Proteins are separated by gel electrophoresis or SDS-polyacrylamide gel electrophoresis (also known as SDS-PAGE), which separates proteins by size. Proteins are then transferred to a sheet of nitrocellulose membrane by placing a nitrocellulose membrane on the gel though other types of paper or membranes can be used. The proteins will retain the same pattern of separation they had on the gel.
The blot is incubated with a generic protein (such as milk proteins) to bind to any remaining sticky places on the nitrocellulose. An antibody is then added to the solution which is able to bind to its specific protein. The antibody has an enzyme (e.g. alkaline phosphatase or horseradish peroxidase) or a dye attached to it which cannot be seen at this time.
The location of the antibody is revealed by incubating it with a colourless substrate that the attached enzyme converts to a coloured product that can be seen and photographed.
More sensitive signal-generating methods can be used, such as ECL (enhanced chemoluminescent) by using the substrate that will luminesce when exposed to the reporter on the secondary antibody.
Western blot can also use isotope-labelled primary antibody, which can be detected directly by X-ray film and does not require the secondary antibody.
Western blot has been widely used in analysing and identifying target proteins. The assay can also be used for semi-quantifying protein concentration through measuring signal intensity of each protein band, if a standard curve of the same protein or a protein at similar molecular weight can be established. Western blot has also been used in clinical laboratories for assisting identification of certain antigen proteins.
NORTHERN BLOT
Northern blot technique is a technique analogous to a Southern blot technique but performed on fragments of RNA and remains a standard method for detection and quantitation of mRNA levels. The northern blot technique was developed in 1977 by James Alwine, David Kemp, and George Stark at Stanford University. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot, named after the biologist Edwin Southern. The major difference is that RNA, rather than DNA, is analysed in the northern blot.
The mRNA can be isolated through the use of oligo (dT) cellulose chromatography to maintain only those RNAs with a poly A tail. RNA samples are then separated by gel electrophoresis. Unlike in the Southern blot, formaldehyde is added to the gel that acts as a denaturant to agarose. For polyacrylamide, urea is the denaturant.
The RNA samples, now separated by size, are transferred to a nylon membrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge is the most effective for use in northern blotting since the negatively charged nucleic acids have a high affinity for them. Once the RNA has been transferred to the membrane it is immobilized through covalent linkage to the membrane by UV light or heat.
After a probe has been labelled, it is hybridized to the RNA on the membrane. The membrane is washed to ensure that the probe has bound specifically and to avoid background signals from arising. The hybrid signals are then detected by X-ray film and can be quantified by densitometry. As in the Southern blot, the hybridization probe may be made from DNA or RNA.
SOUTHERN BLOT
Southern blot technique, named after its inventor the British biologist Edwin M. Southern of Edinburgh University (1975), is a technique for transferring DNA fragments separated by electrophoresis onto a filter, on which specific fragments can then be detected by their hybridization to defined probes.
DNA is digested with a restriction enzyme and separated by gel electrophoresis, usually an agarose gel. The DNA is denatured into single strands by incubation with NaOH.
The DNA is transferred to a membrane which is a sheet of special blotting paper. The DNA fragments retain the same pattern of separation as they had on the gel.
The blot is incubated with many copies of the probe which is single-stranded DNA. This probe will form base pairs with its complementary DNA sequences and bind to form a double-stranded DNA molecule. The probe cannot be seen but it is either radioactive or has an enzyme bound to it (e.g. alkaline phosphatase or horseradish peroxidase).
The location of the probe is revealed by incubating it with a colourless substrate that the attached enzyme converts to a coloured product that can be seen or gives off light or will expose X-ray film. If the probe was labelled with radioactivity, it can expose X-ray film directly.
SOUTHWESTERN BLOT
South-western blot technique is a technique analogous to a Southern blot technique but in which proteins are separated by electrophoresis, transferred to a filter, and probed with DNA fragments to identify expression of specific DNA binding proteins. First described by B. Bowen and colleagues in 1980, it is a lab technique which involves identifying and characterizing DNA-binding proteins by their ability to bind to specific oligonucleotide probes.
Southwestern blotting is also used to investigate DNA-protein interactions. The advantage of this technique over other related methods such as electrophoretic mobility shift assay (EMSA) and DNA foot printing is that it provides information regarding the molecular weight of unknown protein factor. This method combines the features of Southern and Western blotting techniques in which a denaturing SDS-PAGE is first employed to separate proteins electrophoretically by size, and after transferring the proteins to a membrane support, the membrane-bound proteins are renatured and incubated with a (32)P-labelled double-stranded oligonucleotide probe of specific DNA sequence. The interaction of the probe with the protein is later visualized by autoradiography.