Southwestern blot: Difference between revisions
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'''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,<ref name="pmid6243775">{{cite journal |author=Bowen B, Steinberg J, Laemmli UK, Weintraub H |title=The detection of DNA-binding proteins by protein blotting |journal=Nucleic Acids Res. |volume=8 |issue=1 |pages=1–20 |year=1980 |month=January |pmid=6243775 |pmc=327239 |doi= 10.1093/nar/8.1.1|url=http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6243775}}</ref> is a lab technique which involves identifying and characterizing DNA-binding proteins ([[protein]]s that bind to [[DNA]])<ref>{{MeshName|Southwestern+Blot}}</ref> by their ability to bind to specific oligonucleotide probes. The proteins |
'''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,<ref name="pmid6243775">{{cite journal |author=Bowen B, Steinberg J, Laemmli UK, Weintraub H |title=The detection of DNA-binding proteins by protein blotting |journal=Nucleic Acids Res. |volume=8 |issue=1 |pages=1–20 |year=1980 |month=January |pmid=6243775 |pmc=327239 |doi= 10.1093/nar/8.1.1|url=http://nar.oxfordjournals.org/cgi/pmidlookup?view=long&pmid=6243775}}</ref> is a lab technique which involves identifying and characterizing DNA-binding proteins ([[protein]]s that bind to [[DNA]])<ref>{{MeshName|Southwestern+Blot}}</ref> 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. |
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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]]. |
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]]. |
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However, since the first southwestern blottings, many more have been proposed and discovered. |
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 [[Chemical decomposition|degradation]] while being isolated. |
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"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 |
"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 ([[Polyacrylamide gel electrophoresis|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. |
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==References== |
==References== |
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Revision as of 15:02, 18 March 2011
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,[1] is a lab technique which involves identifying and characterizing DNA-binding proteins (proteins that bind to DNA)[2] 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 proteins 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 enzymes 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.
References
- ^ Bowen B, Steinberg J, Laemmli UK, Weintraub H (1980). "The detection of DNA-binding proteins by protein blotting". Nucleic Acids Res. 8 (1): 1–20. doi:10.1093/nar/8.1.1. PMC 327239. PMID 6243775.
{{cite journal}}: Unknown parameter|month=ignored (help)CS1 maint: multiple names: authors list (link) - ^ Southwestern+Blot at the U.S. National Library of Medicine Medical Subject Headings (MeSH)
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