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Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in vitro. Here we describe a simple method for EMSA with fluorescent dye-bound oligo DNA probes and recombinant protein expressed in bacterial cells. Using fluorescent dye instead of radioisotope enables easy handling and long-term storage of labelled-probes without reduction of detection sensitivity.

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Electrophoresis Mobility Shift Assay

Molecular Biology > DNA > DNA-protein interaction
Authors: Masaru Nakata
Masaru NakataAffiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
Bio-protocol author page: a1283
 and Masaru Ohme-Takagi
Masaru Ohme-TakagiAffiliation 1: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan
Affiliation 2: Institute for Environmental Science and Technology, Saitama University, Saitama, Japan
For correspondence: m-takagi@aist.go.jp
Bio-protocol author page: a1284
Vol 4, Iss 7, 4/5/2014, 3686 views, 0 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.1099

[Abstract] Protein (transcription factors and/or transcription cofactors)-binding to DNA is a critical event in regulation of transcription. Electrophoresis Mobility Shift Assay (EMSA), also known as gel shift assay, is a useful tool to detect protein- or protein complex-DNA/RNA interaction and to evaluate DNA binding specificity of transcription factors in vitro. Here we describe a simple method for EMSA with fluorescent dye-bound oligo DNA probes and recombinant protein expressed in bacterial cells. Using fluorescent dye instead of radioisotope enables easy handling and long-term storage of labelled-probes without reduction of detection sensitivity.

Materials and Reagents

  1. Oligo DNA 5’ end-labeled with IRDye 700 or IRDye 800 (sense strand) (Integrated DNA Technologies)
  2. Non-labelled oligo DNA (both sense and antisense strands)
  3. Non-labelled mutated oligo DNA (both sense and antisense strands)
  4. Recombinant DNA-binding proteins expressed in Escherichia coli (E. coli) (10 ng/µl in protein storage buffer)
  5. Sterile distilled water (SDW)
  6. Odyssey infrared EMSA kit (LI-COR, catalog number: 829-07910)
  7. Poly(dI-dC) (Sigma-Aldrich, catalog number: 4929)
  8. Tris
  9. Boric acid
  10. EDTA 2Na
  11. NaCl
  12. HCl
  13. Acrylamide
  14. N,N’-Methylene-bisacrylamide
  15. Glycerol
  16. Triton X-100
  17. Phenylmethylsulfonyl fluoride (PMSF)
  18. β-mercaptoethanol
  19. Ammonium persulfate (APS)
  20. N,N,N',N'-tetramethylethylenediamine (TEMED)
  21. 10x TBE( see Recipes)
  22. 4% native polyacrylamide gel (see Recipes)
  23. Native-PAGE running buffer (see Recipes)
  24. Protein storage buffer (see Recipes)

Equipment

  1. Odyssey CLx Infrared Imaging System (LI-COR)
  2. A set of devices for polyacrylamide gel electrophoresis
  3. Power supply
  4. Refrigerator or cold room
  5. Heat block

Procedure

  1. Suspend lyophilized oligo DNA with dH2O and mix them as follows (final concentration is 50 µM each).
    Probe: labelled and complementary non-labelled oligo DNAs
    Competitor: non-labelled and complementary non-labelled oligo DNAs
    Mutated competitor: mutated non-labelled and complementary non-labelled oligo DNAs
  2. Heat at 100 °C for 10 min on heat block for denature.
  3. Turn off heat block and leave denatured DNAs on the block until room temperature.
  4. Dilute probes to 50 nM with dH2O.
  5. Prepare 4% native polyacrylamide gel and 0.5x TBE.
  6. Pre-electrophoresis for 30 min at 150 V at 4 °C.
  7. Prepare reaction mixtures as follows during pre-electrophoresis:

    Negative Control
    Probe
    Competitor
    Mutated competitor
    Protein (10 ng/µl)
    -
    1 µl
    1 µl
    1 µl
    Probe (50 nM)
    1 µl
    1 µl
    1 µl
    1 µl
    Competitor (50 µM)
    -
    -
    1 µl
    -
    Mutated competitor (50 µM)
    -
    -
    -
    1 µl
    10 x buffer (tube 1)
    2 µl
    2 µl
    2 µl
    2 µl
    25 mM DTT/2.5% Tween-20 (tube 2)
    2 µl
    2 µl
    2 µl
    2 µl
    1 µg/µl Poly (dIdC) (tube 3)
    1 µl
    1 µl
    1 µl
    1 µl
    50% Glycerol (tube 5)
    2 µl
    2 µl
    2 µl
    2 µl
    100 mM MgCl2 (tube 8)
    1 µl
    1 µl
    1 µl
    1 µl
    dH2O
    11 µl
    10 µl
    9 µl
    9 µl
    Note: Tube numbers indicate the vial numbers in Odyssey Infrared EMSA kit.
  8. Place at room temperature for 20 min in dark.
    Note: Avoid light during reaction not to reduce signal intensity.
  9. Add 2 µl of 10x Orange Dye (tube 10) to reaction mixture after reaction.
  10. Wash gel wells with electrophoresis buffer (0.5x TBE) after pre-electrophoresis.
  11. Load the samples on gel and run the gel at 150 V for 2.5 h at 4 °C in dark until the Orange Dye migrates to the bottom of the gel.
  12. Remove gel from glass plate and place it directly on Odyssey.
  13. Adjust focus offset of Odyssey to 1/2 of gel thickness and scan.
    Note: Carefully remove air bubbles between gel and Odyssey.
    Look at supplemental Figure 19 of Reference 1 as a representative EMSA result.

Recipes

  1. 10x TBE
    Consisting of 890 mM Tris, 890 mM boric acid and 20 mM EDTA 2Na (pH 8.3)
    Mix:
    108 g of Tris base
    55 g of boric acid
    3.7 g of EDTA 2Na
    Add dH2O to 1 L
    Autoclave and stored at room temperature
  2. 4% native polyacrylamide gel
    Consisting of 4% acrylamide, 0.5x TBE, 2.5% glycerol, 0.1% APS, 0.1% TEMED
    Mix:
    2.67 ml of 30% acrylamide (acrylamide: bisacrylamide = 29:1)
    1 ml of 10x TBE
    1 ml of 50% glycerol
    0.2 ml of 10% APS
    20 µl of TEMED
    Add dH2O to 20 ml
  3. Native-PAGE running buffer
    0.5x TBE
  4. Protein storage buffer
    Consisting of 10 mM Tris-HCl (pH7.5), 150 mM NaCl, 0.1% Triton X-100, 1 mM PMSF, 0.05% β-mercaptoethanol, 50% glycerol
    Mix:
    1 ml of 1 M Tris-HCl (pH 7.5)
    3 ml of 5 M NaCl
    1 ml of 10% Triton X-100
    1 ml of 100 mM PMSF
    50 µl of β-mercaptoethanol
    50 g of glycerol
    Add dH2O to 100 ml

Acknowledgments

This protocol is adapted from Nakata et al. (2013).

References

  1. Nakata, M., Mitsuda, N., Herde, M., Koo, A. J., Moreno, J. E., Suzuki, K., Howe, G. A. and Ohme-Takagi, M. (2013). A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell 25(5): 1641-1656.


How to cite this protocol: Nakata, M. and Ohme-Takagi, M. (2014). Electrophoresis Mobility Shift Assay. Bio-protocol 4(7): e1099. DOI: 10.21769/BioProtoc.1099; Full Text



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