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An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. If the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to the protein, which is 'shifted' up on the gel (since it has moved more slowly). Here, a protocol to carry out an EMSA assay is described.

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[Bio101] A General EMSA (Gel-shift) Protocol

Molecular Biology > DNA > DNA-protein interaction
Author: Ran Chen
Ran ChenAffiliation: Department of Genetics, Stanford University, Stanford, USA
For correspondence: rcchen@jfkbio.com
Bio-protocol author page: a34
2/5/2011, 22171 views, 3 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.24

[Abstract] An electrophoretic mobility shift assay (EMSA), also referred to as mobility shift electrophoresis, a gel shift assay, gel mobility shift assay, band shift assay, or gel retardation assay, is a common technique used to study protein-DNA or protein-RNA interactions. The control lane (the DNA/RNA probe without protein present) will contain a single band corresponding to the unbound DNA or RNA fragment. If the protein is capable of binding to the fragment, the lane with protein present will contain another band that represents the larger, less mobile complex of nucleic acid probe bound to the protein, which is 'shifted' up on the gel (since it has moved more slowly). Here, a protocol to carry out an EMSA assay is described.

Keywords: EMSA, Gel-shift, Binding

Materials and Reagents

  1. DTT (Promega Corporation, catalog number: V3151)
  2. Poly-dIdC (Sigma-Aldrich, catalog number: P4929-10UN)
  3. 32P-labeled probe
    Note: Oligo DNA probe can be synthesized ordered from IDT, a DNA synthesis company, then labeled by yourself.
  4. BSA (Sigma-Aldrich, catalog number: 05470-5G)
  5. General chemicals (Sigma-Aldrich)
  6. 1.5x binding buffer (see Recipes)
  7. 10x TBE buffer (see Recipes)

Equipment

  1. Plates
  2. Spacers
  3. Clamps
  4. Saran wrap
  5. Whatman paper (GE Healthcare)

Procedure

  1. Pour protein polyacrylamide gel.
    1. Assemble plates, spacers, and clamps. Seal with 1% agarose to prevent leaks.
    2. Pour 5% polyacrylamide gel.
      Plate size
      Large
      Medium
      H2O
      78 ml
      39 ml
      10x TBE
      5 ml
      2.5 ml
      30% acrylamide stock (19:1)
      16.6 ml
      8.4 ml
      10% APS
      1,000 μl
      500 μl
      Mix well while minimizing bubble formation. Add 100 μl/50 μl TEMED. Mix and pour, add combs. Gel will take ~10 min to polymerize. After polymerization, gel can be wrapped in saran wrap and stored at 4 °C.
  2. Prepare 5x binding buffer.
  3. Set binding reaction:
    1 μl of poly-dIdC (1 μg/μl in TE)
    2 μl of 5x binding buffer
    1 μl of labeled probe
    1 μl cold competitor - unlabeled DNA fragments containing the binding sequences (if needed)
    0.1 μl 100x BSA
    X μl nuclear extract (5 μg protein total)
    Add H2O to 10 μl final volume
    Incubate for 30 min at room temperature (RT). Add antibody for supershift (if needed). Incubate additional 30 min at RT.
  4. While binding reaction is incubating, run the polyacrylamide gel without any sample at 150 V, 30 min, using 0.5x TBE as the running buffer. Then run samples on the polyacrylamide gel for ~2 h at 150 V.
  5. Dry the gel (optional).
    Transfer gel to Whatman paper. Cover top of gel with saran wrap and dry at 80 °C in vacuum dryer for 1-2 h.
  6. Expose the gel.
    Place gel in cassette with reflection screen. Add film and place in -80 °C freezer.

Recipes

  1. 5x binding buffer
    Composition
    Recipe for 10 ml
    50 mM Tris HCl (pH 8.0)
    0.5 ml of 1 M Tris HCl (pH 8.0)
    750 mM KCl
    3 ml of 2.5 M KCl
    2.5 mM EDTA
    50 μl of 0.5 M EDTA (pH 8.0)
    0.5% Triton-X 100
    50 μl Triton-X 100
    62.5 % glycerol (v/v)
    7.87 g glycerol
    1 mM DTT
    add DTT fresh before use
  2. 10x TBE buffer (1 L)
    106 g of Tris base
    55 g of boric acid
    40 ml of 0.5 M EDTA (pH 8.0)

Acknowledgments

This work was funded by 5050 project by Hangzhou Hi-Tech District, Funding for Oversea Returnee by Hangzhou City, ZJ1000 project by Zhejiang Province. The protocol was developed in the Cohen Lab, Department of Genetics, Stanford University, CA, USA.

References

  1. Chen, R., Liliental, J. E., Kowalski, P. E., Lu, Q. and Cohen, S. N. (2011). Regulation of transcription of hypoxia-inducible factor-1alpha (HIF-1alpha) by heat shock factors HSF2 and HSF4. Oncogene 30(22): 2570-2580.


How to cite this protocol: Chen, R. (2011). A General EMSA (Gel-shift) Protocol. Bio-protocol Bio101: e24. DOI: 10.21769/BioProtoc.24; Full Text



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1/8/2013 2:39:16 PM  

hayden thain
flinders uni

Hi I wanted to ask if its possible to use gel retardation on Sigma B as i believe it is the regulator of my target gene in S.aureus, or if there is a better protocol or method?

Thanks

1/10/2013 11:09:17 PM  

RAN CHEN
Stanford

Gel retardation is a good option if you know the candidate binding sites in the promoter region of your target gene. In your case, my opinion is that Ch-IP could be better choice.

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1/24/2012 5:58:09 AM  

Lin Fang
Stanford University

Hello, this is Lin Fang, associated editor of bio-protocol.org. The following is a organized answer to your question of our editorial board and the author Ran Chen.

Reporter assay in cells and Electric Mobility Shift Assay (EMSA) are often utilized to address the interaction between transcription factors and promoters. Reporter assay in cells is normally done first as it is often easier, quicker and more reliable in terms of whether transcription factors are involved in regulating promoter activities. EMSA can provide more details such as whether two transcription factors are present in two or the same complex. However, it is more tricky and prone to false results and requires high resolution, so often done after the reporter assays in cells. If interaction between A and B want to be assayed directly, Coimmunoprecipitation, GST-Pulldown and FRET (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2937893/?tool=pubmed) can be performed.

Dual Luciferase Reporter Assays (Protocol is coming soon! )

Transfect cells respectively with the following combinations I-IV:
? I: pRL-CMV+ pGL3-G
? II: pRL-CMV+ pGL3-G + pA
? III: pRL-CMV + pGL3-G + pB
? IV: pRL-CMV + pGL3-G + pA + pB

? pRL-CMV: Promega E2261. pGL3: Promega E1751. pGL3-p: pGL3 reporter construct driven by promoter G. pA and pB: Transcription factor A and B in your favorite expression vector.
? Control groups I’-IV’ should also be included except that pGL3-G in corresponding group A-D is replaced by pGL3 which is a promoterless reporter constructs.

Analyze the transfection with Dual Luciferase Assay (Promega E1910). Expected results are
? Here, pGL3-G should be much stronger than pGL3 (5 fold) to confirm true activation.
? If addition of A and/or B change the promoter activity of G, A and/or B bind to promoter G.
? If addition of both A and B show additive effect of addition of A and B alone, it suggests that A and B MAY NOT bind each other.
? If addition of both A and B show synergistic effect of addition of A and B alone, it suggests that A and B MAY bind each other.
To further delineate the binding sites of A and B on promoter G, potential binding sites of A and B can be predicted according to their canonical binding sequences and mutated. The effect of mutations in promoter G can be assayed with the reporter assay.
If high activation pGL3-G in the absence of A or B, siRNA of A or B can be tested too.

EMSA in the presence of competing DNA sequences and antibodies against A and B respectively
Before proceeding, it is desirable to narrow the labeled DNA sequences as well as non-labeled competing DNA sequences to shorter length. So it is better to narrow down the sequence by footprinting and identification of potential binding sites of A and B referring to canonical A and B binding sites.
Material:
? Cell nuclear extract (make sure it contains A and B)
? Labeled DNA sequence
? Unlabeled DNA sequence
? A antibody
? B antibody
? Non-immune serum
Native-PAGE gel
Mix in the following combinations
1. Labeled DNA
2. Labeled DNA + Cell nuclear extract
3. Labeled DNA + Cell nuclear extract + unlabeled DNA
4. Labeled DNA + Cell nuclear extract + A antibody
5. Labeled DNA + Cell nuclear extract + B antibody
6. Labeled DNA + Cell nuclear extract + non-immune serum
Run the reaction in native PAGE gel

Expected results:
? Labeled DNA + Cell nuclear extract should form several bands, containing specific and nonspecific complex.
? Bands representing specific complex should disappear in the presence of unlabeled DNA.
? If transcription factor A or B is present in the complexes, their corresponding antibody should supershif the specific complex, aka, running slower in the PAGE gel.
? If A and B antibody supershift the same band, A and B form one complex.
? Non-immune serum should not supershift the specific complex.






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1/17/2012 2:16:15 PM  

Please design a protocal to identify whether transcriptional factor A and transcriptional factor B may bind together to form a complex, and bind to the promoter of G gene to increase its transcriptional level. (Please use the folowing techniqes: Yeast two-hibrid, ChIP, EMSA, Reporter gene assay)

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