Overview: Gel Shift, or Band Shift Assay, or Electrophoretic Mobility Shift Assay (EMSA) is a technique for studying gene regulation and determining protein:DNA interactions. The assay is based on the observation that complexes of protein and DNA migrate through a non-denaturing polyacrylamide gel more slowly than free DNA fragments or double-stranded oligonucleotides. The gel shift assay is carried out by first incubating a protein(s) (such as nuclear or cell extract) with a 32P end-labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest or other unrelated DNA sequences.
Related Categories:
Protocols
- Aimee's non-Radioactive EMSA Protocol
Well written non-radioactive EMSA methods with description of the entire procedure including labelling of the oligos and nuclear extraction.
Added: Sat Feb 26 2005, Reviews: 0 Write review - EMSA Protocol (Jonathan Flint Lab, Wellcome Trust Centre for Human Genetics)
The EMSA, or Electrophoretic Mobility Shift Assay, is a method for studying DNA-protein interactions in vitro. This protocol gives detailed procedures on primer design, primer annealing, primer labelling, probe purification, making acrylamide gel, carring out binding reaction, loading and drying and photographing of gel.
http://www.well.ox.ac.uk/flint/EMSA.htm
Added: Wed Jun 14 2006, Hits: 8895, Reviews: 0 Write review Cached - Gel Mobility Shift Assay for transcription factor binding (Contributed by Riddhish Shah, Hueley Lab)
Protocol can be used to investigate binding of transcription factor to your DNA seq. We use to see the differences due to polymorphic spot.
Added: Tue Apr 08 2003, Reviews: 0 Write review -
Band Shift Analysis
(Dario Neri's labs, Institute of Pharmaceutical Sciences ETHZ)
The procedure is for the determination of affinity constants and kinetic dissociation constants by band-shift assay refers to an ideal antibody fragment (e.g., a scFv or an Fab fragment) binding to a well-behaved protein antigen (pure, of well-defined oligomeric state, migrating as a single band in non-denaturing gel electrophoresis.
http://www.pharma.ethz.ch/institute_groups/biomacr...
Added: Mon Sep 09 2002, Hits: 4498, Reviews: 0 Write review Cached -
Band shifting
(Promega)
The gel shift assay is performed by incubating a purified protein, or a complex mixture of proteins (such as nuclear or cell extract preparations), with a 32P end-labeled DNA fragment containing the putative protein binding site. The reaction products are then analyzed on a nondenaturing polyacrylamide gel. The specificity of the DNA-binding protein for the putative binding site is established by competition experiments using DNA fragments or oligonucleotides containing a binding site for the protein of interest, or other unrelated DNA sequences.
http://www.promega.com/tbs/tb110/tb110.html
Added: Tue May 14 2002, Hits: 8302, Reviews: 0 Write review Cached -
Bank Shift Assay for Kd Determination
(Neri's Lab, Institute of Pharmaceutical Sciences ETHZ)
The procedure is for the determination of affinity constants and kinetic dissociation constants by band-shift assay refers to an ideal antibody fragment (e.g., a scFv or an Fab fragment) binding to a well-behaved protein antigen (pure, of well-defined oligomeric state, migrating as a single band in non-denaturing gel electrophoresis. As mentioned in the introduction, either the antibody or the antigen has to be labeled in these assays.
http://www.pharma.ethz.ch/institute_groups/biomacr...
Added: Tue May 14 2002, Hits: 2872, Reviews: 0 Write review Cached -
Electrophoretic Mobility Shift Assay (EMSA)
(Sugden Laboratory, McArdle Laboratory for Cancer Research)
20cm Gel Preparation, Sample Separation, and Visualization
http://mcardle.oncology.wisc.edu/sugden/protocols/...
Added: Sat Mar 01 2008, Hits: 1169, Reviews: 0 Write review -
Electrophoretic Mobility Shift Assay (EMSA)
(Sugden Laboratory, McArdle Laboratory for Cancer Research)
http://mcardle.oncology.wisc.edu/sugden/protocols/...
Added: Sat Mar 01 2008, Hits: 1001, Reviews: 0 Write review -
Electrophoretic Mobility Shift Assay (EMSA) for the Detection of Nuclear NF-kB
(CellDeath.de)
Nuclear extracts are incubated with a radioactive labeled oligonucleotide probe which contains the specific recognition sequence for NF-kB. The binding reaction occurs under specific salt/pH conditions in a binding buffer. Poly-dIdC is added to prevent unspecific binding of proteins to the NF-kB oligonucleotide probe and by this to reduce background. After binding, the samples are separated on a non-denaturating PAGE gel and bands are detected by autoradiography.
http://www.celldeath.de/apometh/emsa.html
Added: Mon Mar 05 2007, Hits: 2158, Reviews: 0 Write review Cached -
EMSA Using Oligos
(Mike A. Dyer)
Anneal two complementary oligos to make ds oligos as the probe for EMSA assay.
http://axon.med.harvard.edu/~cepko/protocol/mike/E...
Added: Tue May 14 2002, Hits: 6395, Reviews: 0 Write review Cached