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The interaction of transcriptional or co-transcriptional factors with DNA is crucial for changes of neuronal gene expression during normal brain development as well as neurodegeneration. The electrophoretic mobility shift assay (EMSA) is a very powerful technique for studying changes of neuronal gene expression and determining protein: DNA interactions. EMSA can be used qualitatively to identify specific transcriptional or co-transcriptional factors in brain crude lysates or primary neurons and, in conjunction with mutagenesis, to identify the important binding sequences within a given gene. An advantage of studying protein: DNA interaction by an electrophoretic assay provides a better understanding of epigenetic changes during normal brain development and neurodegenerative process.

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A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron

Neuroscience > Development > Neuron
Author: Jiali Li
Jiali LiAffiliation: Department of Cell Biology and Neuroscience, Nelson Biological Laboratories, Rutgers University, Piscataway, NJ, USA
For correspondence: jli@dls.rutgers.edu
Bio-protocol author page: a179
Vol 2, Iss 23, 12/5/2012, 8995 views, 3 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.300

[Abstract] The interaction of transcriptional or co-transcriptional factors with DNA is crucial for changes of neuronal gene expression during normal brain development as well as neurodegeneration. The electrophoretic mobility shift assay (EMSA) is a very powerful technique for studying changes of neuronal gene expression and determining protein: DNA interactions. EMSA can be used qualitatively to identify specific transcriptional or co-transcriptional factors in brain crude lysates or primary neurons and, in conjunction with mutagenesis, to identify the important binding sequences within a given gene. An advantage of studying protein: DNA interaction by an electrophoretic assay provides a better understanding of epigenetic changes during normal brain development and neurodegenerative process.

Materials and Reagents

  1. Neuronal cell pellet
  2. Biotin 5' end-labeled and non-labeled DNA probes (competition) (Integrated DNA Technologies)
    Table S- sequences of EMSA probes


  3. Positively charged nylon membrane (Sigma-Aldrich, catalog number: Z670197)
  4. Tris base
  5. Boric acid
  6. EDTA
  7. BSA
  8. Poly (dIdC) (0.5 μg/μl) (Pierce Antibodies, catalog number: 20148)
  9. Antibody (ab1437)
  10. 5x loading buffer (QIAGEN, catalog number: 1037650)
  11. X-ray film
  12. High-quality blotting paper (Whatman, catalog number: 3030-931)
  13. Polyacrylamide gel in 0.5x TBE
  14. Cytoplasmic extract buffer (NE-PER Nuclear and Cytoplasmic Extraction Kit) (Pierce Antibodies, catalog number: 78835)
  15. Washing buffer (LightShift Chemiluminescent EMSA Kit) (Pierce Antibodies, catalog number: 20148)
  16. Nuclear extraction buffer (NE-PER Nuclear and Cytoplasmic Extraction Kit) (Pierce Antibodies, catalog number: 78835)
  17. 2x reaction buffer (LightShift Chemiluminescent EMSA Kit) (Pierce Antibodies, catalog number: 20148)
  18. Acrylamide
  19. Bis-acrylamide
  20. TBE buffer
  21. TEMED
  22. Ammonium persufate
  23. Phosphatase inhibitors
  24. 6% non-denature PAGE gel (see Recipes)
  25. 5x TBE (pH 8.3) (see Recipes)

Equipment

  1. Centrifuges
  2. UV lamp or crosslinking device equipped with 254 nm bulbs or 312 nm transilluminator
  3. Electrophoresis apparatus
  4. Electroblotter or capillary transfer apparatus
  5. 1.5 ml microcentrifuge tube

Procedure

  1. Prepare the nuclear protein extract from neuron
    1. Collect 1-5 x 106-7 neurons pellet in 1.5 ml microcentrifuge tube (DIV 14 Primary cultural neuron was from mouse E16.5 cortex).
    2. Resuspend neurons in 200 μl cytoplasmic extract buffer with protease and phosphatase inhibitors, and keep on ice for 10 min to break the cell membrane.
    3. Spin cells at 16,000 x g for 5 min at 4 °C to separate nuclei with cytoplasmic component (quality of fraction was tested by nuclear/cytoplasmic protein markers-HDAC1 and HSP90).
    4. Remove supernatant as cytoplasmic extract.
    5. Wash the nuclear pellet with 300 μl washing buffer.
    6. Spin cells at 16,000 x g for 10 min at 4 °C to pellet the nuclei.
    7. Resuspend the nuclear pellet in 100 μl nuclear extraction buffer and aliquot the lysate into10 μl/tube.
    8. Freeze the nuclear extracts in -80 °C.
    9. Measure protein concentration and adjust it to 1 μg/μl with the extraction buffer for use in gel shift assay.

  2. Prepare and pre-run gel
    1. Prepare a native PAGE gel in 0.5x TBE. The appropriate polyacrylamide percent depends on the size of the target DNA and the binding protein. Most systems use a 4 -6% PAGE gel in 0.5x TBE.
    2. Place the gel in the electrophoresis unit. Fill the inner chamber with 0.5x TBE to a height several millimeters above the top of the wells. Fill the outside of the tank with 0.5x TBE to just above the bottom of the wells, which reduces heat during electrophoresis. Flush wells and pre-electrophorese the gel for 30-60 min at 100 V.

  3. Perform binding reactions
    1. 2x reaction buffer 12 μl.
    2. BSA (1 μg/μl) 3 μl.
    3. Poly (dIdC) (0.5 μg/μl) 2 μl.
    4. Nuclear extract (1 μg/μl) 3 μl (cytoplasmic extract as control).
    5. dH2O 3 μl.
    6. Keep at room temperature or on ice for 10 min without Antibody, 20 min with Antibody; Keep rotation (option).
    7. Add in Biotin-labeled DNA probe (20 fmol/ reaction).
    8. Keep at room temperature for 20 min.

  4. Electrophorese binding reactions
    1. Add 5 μl of 5x loading buffer to each 20 μl binding reaction, pipetting up and down several times to mix. Run gel at 200 V for 1-1.5 h. Use DNA loading buffer in lane 1 as indicator of free probe. Free probe usually run at the same mobility as the blue dye of the DNA loading buffer. Stop the gel when the dye runs at 3 cm to the bottom.

  5. Electrophoretic transfer of binding reactions to nylon membrane
    1. Soak nylon membrane in 0.5x TBE for 15 min.
    2. Sandwich the gel, nylon membrane and blotting paper in a clean electrophoretic transfer unit.
    3. Transfer at 380 mA (~100 V) for 30 min.
    4. When the transfer is complete, place the on a dry paper towel for 1-3 min.

  6. Crosslink transferred DNA to membrane and detection
    1. 10-15 min with the membrane face down on a transilluminator equipped with 312 nm bulbs.
    2. After the membrane is crosslinked, proceed directly to the LightShift Chemiluminescent detection kit. Alternatively, the membrane may be stored dry at room temperature for several days.

Recipes

  1. 6% non-denature PAGE gel
    For example, to make 50 ml volumes gel:
    7.5ml 40% acrylamide
    5ml 2% Bis-acrylamide
    2.5ml 10x TBE buffer
    50 μl TEMED
    0.5 ml 10% ammonium persufate
    34.5 ml dH2O
  2. 5x TBE (pH 8.3)
    450 mM Tris
    450 mM boric acid
    10 mM EDTA

Acknowledgments

This protocol is adapted from Li et al. (2012).

References

  1. Li, J., Chen, J., Ricupero, C. L., Hart, R. P., Schwartz, M. S., Kusnecov, A. and Herrup, K. (2012). Nuclear accumulation of HDAC4 in ATM deficiency promotes neurodegeneration in ataxia telangiectasia. Nat Med 18(5): 783-790.


How to cite this protocol: Li, J. (2012). A Protocol for Electrophoretic Mobility Shift Assay (EMSA) from Primary Neuron. Bio-protocol 2(23): e300. DOI: 10.21769/BioProtoc.300; Full Text



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7/27/2015 4:33:30 PM  

Britney Helling
UC Denver

Hi,
What is the purpose of the crosslinking and can I get away with not doing it if I image it directly after chemiluminescence?
Thanks

Reply

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9/5/2014 10:15:26 AM  

Randi Foxall
University of New Hampshire

I am trying to troubleshoot an EMSA and came across your protocol. I am performing an EMSA using 5' biotin ssDNA, not dsDNA as in your protocol. Do you know if a ssDNA probe should work with the detection assays for Biotin?

9/6/2014 12:00:39 AM  

Jiali Li (Author)
Department of Cell Biology and Neuroscience, Nelson Biological Laboratories,Rutgers University

It should work too if it binds to your target efficiently.

Reply

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7/19/2013 5:08:48 AM  

pritha ray
ACTREC, Tata memorial Centre

Hi,
Thanks for the protocol. In my experiment i am not seeing any trace of the free labeled probe at the bottom of the gel though a retarded band is present at high molecular weight. This band can be competed out with excess cold probe. I tried running a very short gel by keeping bromophenol blue at 1/3rd of the gel and still didnot see any free probe. The size of the oligo is 50 bp. Please suggest.

7/19/2013 6:21:22 AM  

Jiali Li (Author)
Department of Cell Biology and Neuroscience, Nelson Biological Laboratories,Rutgers University

Please make sure whether a correct DNA-running gel is used. Is any positive control set up?

Reply

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