Southwestern blot
Encyclopedia
Southwestern blotting, based along the lines of Southern blotting (which was created by Edwin Southern
) and first described by B. Bowen and colleagues in 1980, is a lab technique which involves identifying and characterizing DNA-binding proteins (protein
s that bind to DNA
) by their ability to bind to specific oligonucleotide probes. The proteins are separated by gel electrophoresis
and are subsequently transferred to nitrocellulose
membranes similar to other types of blotting.
The name southwestern blotting is based on the fact that this technique detects DNA-binding proteins, since DNA detection is by Southern blotting and protein detection is by western blotting.
However, since the first southwestern blottings, many more have been proposed and discovered. The former protocols were hampered by the need for large amounts of protein
s and their susceptibility to degradation
while being isolated.
"Southwestern blot mapping" is performed for rapid characterization of both DNA-binding proteins and their specific sites on genomic DNA. Proteins are separated on a polyacrylamide
gel (PAGE) containing sodium dodecyl sulfate
(SDS), renatured by removing SDS in the presence of urea, and blotted onto nitrocellulose
by diffusion. The genomic DNA region of interest is digested by restriction enzyme
s selected to produce fragments of appropriate but different sizes, which are subsequently end-labeled and allowed to bind to the separated proteins. The specifically bound DNA is eluted from each individual protein-DNA complex and analyzed by polyacrylamide gel electrophoresis. Evidence that tissue-specific DNA binding proteins may be detected by this technique has been presented. Moreover, their sequence-specific binding allows the purification of the corresponding selectively bound DNA fragments and may improve protein-mediated cloning of DNA regulatory sequences.
Edwin Southern
Sir Edwin Mellor Southern, FRS is an English 2005 Lasker Award-winning molecular biologist. His award was for the invention of the Southern blot, now a common laboratory procedure, when he was working at the University of Edinburgh....
) and first described by B. Bowen and colleagues in 1980, is a lab technique which involves identifying and characterizing DNA-binding proteins (protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s that bind to DNA
DNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
) by their ability to bind to specific oligonucleotide probes. The proteins are separated by gel electrophoresis
Gel electrophoresis
Gel electrophoresis is a method used in clinical chemistry to separate proteins by charge and or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge...
and are subsequently transferred to nitrocellulose
Nitrocellulose
Nitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...
membranes similar to other types of blotting.
The name southwestern blotting is based on the fact that this technique detects DNA-binding proteins, since DNA detection is by Southern blotting and protein detection is by western blotting.
However, since the first southwestern blottings, many more have been proposed and discovered. The former protocols were hampered by the need for large amounts of protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s and their susceptibility to degradation
Chemical decomposition
Chemical decomposition, analysis or breakdown is the separation of a chemical compound into elements or simpler compounds. It is sometimes defined as the exact opposite of a chemical synthesis. Chemical decomposition is often an undesired chemical reaction...
while being isolated.
"Southwestern blot mapping" is performed for rapid characterization of both DNA-binding proteins and their specific sites on genomic DNA. Proteins are separated on a polyacrylamide
Polyacrylamide
Polyacrylamide is a polymer formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N-methylenebisacrylamide. Polyacrylamide is not toxic...
gel (PAGE) containing sodium dodecyl sulfate
Sodium dodecyl sulfate
Sodium dodecyl sulfate , sodium laurilsulfate or sodium lauryl sulfate is an organic compound with the formula CH311OSO3Na). It is an anionic surfactant used in many cleaning and hygiene products...
(SDS), renatured by removing SDS in the presence of urea, and blotted onto nitrocellulose
Nitrocellulose
Nitrocellulose is a highly flammable compound formed by nitrating cellulose through exposure to nitric acid or another powerful nitrating agent. When used as a propellant or low-order explosive, it is also known as guncotton...
by diffusion. The genomic DNA region of interest is digested by restriction enzyme
Restriction enzyme
A Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
s selected to produce fragments of appropriate but different sizes, which are subsequently end-labeled and allowed to bind to the separated proteins. The specifically bound DNA is eluted from each individual protein-DNA complex and analyzed by polyacrylamide gel electrophoresis. Evidence that tissue-specific DNA binding proteins may be detected by this technique has been presented. Moreover, their sequence-specific binding allows the purification of the corresponding selectively bound DNA fragments and may improve protein-mediated cloning of DNA regulatory sequences.