Difference between revisions of "Electrophoretic mobility shift assay"

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==Overview==
 
This assay permits testing of specific binding of proteins to DNA fragments.  DNA that is bound to protein will migrate slower in a nondenaturing polyacrylamide gel than unbound DNA during electrophoresis.
 
This assay permits testing of specific binding of proteins to DNA fragments.  DNA that is bound to protein will migrate slower in a nondenaturing polyacrylamide gel than unbound DNA during electrophoresis.
  
 
Most protocols rely on <sup>32</sup>P labelling of the DNA fragment.  However, it is also possible to detect the DNA via nonradioactive detection methods (like fluorescence).  Ethidium bromide staining is generally not sensitive enough since usually small amounts of DNA are used in this assay.
 
Most protocols rely on <sup>32</sup>P labelling of the DNA fragment.  However, it is also possible to detect the DNA via nonradioactive detection methods (like fluorescence).  Ethidium bromide staining is generally not sensitive enough since usually small amounts of DNA are used in this assay.
 
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==Specific Protocols==
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[[Knight:Electrophoretic mobility shift assay]]
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==References==
 
==References==
 
===Background information===
 
===Background information===
*[http://www.piercenet.com/Products/Browse.cfm?fldID=99A16E15-3470-4FD1-9A3C-0BBABBBE45F9 EMSA] by Pierce
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#[http://www.piercenet.com/Products/Browse.cfm?fldID=99A16E15-3470-4FD1-9A3C-0BBABBBE45F9 EMSA] by Pierce
*[[Wikipedia:Electrophoretic mobility shift assay|Electrophoretic mobility shift assay]] from Wikipedia
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#[[Wikipedia:Electrophoretic mobility shift assay|Electrophoretic mobility shift assay]] from Wikipedia
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===Papers===
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<biblio>
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#Garner-NAR-1981 pmid=6269071
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// early paper describing gel shift assays
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#Jing-Electrophoresis-2004 pmid=15300760
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#Jing-Proteomics-2003 pmid=12872218
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</biblio>
  
 
===Protocols===
 
===Protocols===
*[http://www.mrw.interscience.wiley.com/cp/cpmb/articles/mb1202/frame.html Mobility Shift DNA-Binding Assay Using Gel Electrophoresis] from Current Protocols in Molecular Biology
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#[http://www.mrw.interscience.wiley.com/cp/cpmb/articles/mb1202/frame.html Mobility Shift DNA-Binding Assay Using Gel Electrophoresis] from Current Protocols in Molecular Biology
*[http://www.molecularcloning.com/members/protocol.jsp?pronumber=2&chpnumber=17 Gel Retardation Assays for DNA-binding Proteins] from [[Molecular Cloning]]
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#[[doi:10.1101/pdb.prot3948 | Gel Retardation Assays for DNA-binding Proteins]] from [[Molecular Cloning]] (subscription required)
*[http://probes.invitrogen.com/media/pis/mp33075.pdf EMSA kit] from Invitrogen (primary advantage is that this protocol doesn't require use of radioactivity or prelabelling of DNA, sensitivity is questionable though)
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*[http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=06010701 LightShift Chemiluminescent EMSA Kit] from Pierce (claimed to be more sensitive than radioactive and digoxigenin methods)
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==Kits==
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#[http://probes.invitrogen.com/media/pis/mp33075.pdf EMSA kit] from Invitrogen  
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#*primary advantage is that this protocol doesn't require use of radioactivity or prelabelling of DNA, sensitivity is questionable however
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#*Also see relevant papers <cite>Jing-Proteomics-2003, Jing-Electrophoresis-2004</cite>.
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#[http://www.piercenet.com/Objects/View.cfm?type=ProductFamily&ID=06010701 LightShift Chemiluminescent EMSA Kit] from Pierce  
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#*claimed to be more sensitive than radioactive and digoxigenin methods
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[[Category:Protocol]]
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[[Category:In vitro]]
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[[Category:Protein]]
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[[Category:DNA]]

Latest revision as of 07:44, 15 April 2008

Overview

This assay permits testing of specific binding of proteins to DNA fragments. DNA that is bound to protein will migrate slower in a nondenaturing polyacrylamide gel than unbound DNA during electrophoresis.

Most protocols rely on 32P labelling of the DNA fragment. However, it is also possible to detect the DNA via nonradioactive detection methods (like fluorescence). Ethidium bromide staining is generally not sensitive enough since usually small amounts of DNA are used in this assay.

Specific Protocols

Knight:Electrophoretic mobility shift assay

References

Background information

  1. EMSA by Pierce
  2. Electrophoretic mobility shift assay from Wikipedia

Papers

  1. Garner MM and Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047-60. PubMed ID:6269071 | HubMed [Garner-NAR-1981]
    early paper describing gel shift assays
  2. Jing D, Beechem JM, and Patton WF. The utility of a two-color fluorescence electrophoretic mobility shift assay procedure for the analysis of DNA replication complexes. Electrophoresis. 2004 Aug;25(15):2439-46. DOI:10.1002/elps.200405994 | PubMed ID:15300760 | HubMed [Jing-Electrophoresis-2004]
  3. Jing D, Agnew J, Patton WF, Hendrickson J, and Beechem JM. A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels. Proteomics. 2003 Jul;3(7):1172-80. DOI:10.1002/pmic.200300438 | PubMed ID:12872218 | HubMed [Jing-Proteomics-2003]
All Medline abstracts: PubMed | HubMed

Protocols

  1. Mobility Shift DNA-Binding Assay Using Gel Electrophoresis from Current Protocols in Molecular Biology
  2. Gel Retardation Assays for DNA-binding Proteins from Molecular Cloning (subscription required)

Kits

  1. EMSA kit from Invitrogen
    • primary advantage is that this protocol doesn't require use of radioactivity or prelabelling of DNA, sensitivity is questionable however
    • Also see relevant papers [2, 3].
  2. LightShift Chemiluminescent EMSA Kit from Pierce
    • claimed to be more sensitive than radioactive and digoxigenin methods