Electrophoretic mobility shift assay

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==Overview==
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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.
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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==
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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
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===Background information===
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*[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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#[http://www.piercenet.com/Products/Browse.cfm?fldID=99A16E15-3470-4FD1-9A3C-0BBABBBE45F9 EMSA] by Pierce
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*[http://probes.invitrogen.com/media/pis/mp33075.pdf EMSA kit] from Invitrogen (primary advantage is that this protocol doesn't require use of radioactivity)
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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>
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===Protocols===
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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
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#[[doi:10.1101/pdb.prot3948 | Gel Retardation Assays for DNA-binding Proteins]] from [[Molecular Cloning]] (subscription required)
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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]]

Current revision

Contents

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. . pmid:6269071. PubMed HubMed [Garner-NAR-1981]
    early paper describing gel shift assays

  2. Jing D, Beechem JM, and Patton WF. . pmid:15300760. PubMed HubMed [Jing-Electrophoresis-2004]
  3. Jing D, Agnew J, Patton WF, Hendrickson J, and Beechem JM. . pmid:12872218. PubMed 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
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