Molecular Techniques and Methods

Gel Mobility Shift Assay for DNA-Binding Proteins
Using High-Ionic-Strength PAGE

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The gel mobility shift assay is based on the observation that DNA-protein complexes migrate through polyacrylamide gels more slowly than unbound DNA fragments. The sensitivity of this assay enables femptomole quantities of DNA-binding proteins to be detected. The use of mobility shift PAGE provides additional information on the number and type of proteins bound. Each distinct species of protein bound to the probe generates a complex of distinct mobility and specificity so that interactions of several proteins binding to a single DNA fragment can be observed. Even if multiple proteins recognize overlapping sites on the DNA fragment, the complexes formed by each can be resolved and characterized. The mobility of a protein-DNA complex through a native (nondenaturing) polyacrylamide gel is determined primarily by the size and charge of the protein bound to the DNA fragment and by the conformation of the protein-DNA complex. The mobility of the protein-DNA complex is only slightly affected by changes in the size of the DNA fragment used as probe. As a result, changing gel conditions such as acrylamide percentage, acrylamide: bisacrylamide ratio, and pH can significantly alter the mobility of a given protein-DNA complex. In some cases this alteration may be sufficient to permit the resolution of two protein-DNA complexes, allowing the DNA-binding specificity of each to be independently and unambiguously ascertained. Use of high-ionic-strength buffer systems can also enhance the resolution of protein-DNA complexes. The higher ionic strength makes recirculation of buffer unnecessary. Moreover, the resulting bands tend to be sharper than those of the low-ionic-strength system, allowing better resolution of protein-DNA complexes. However, although many protein-DNA interactions may survive the high ionic strength of this buffer system, some do not. Therefore, it is helpful to try both low- and high-ionic-strength buffer systems for each putative protein-DNA interaction. With this alternate buffer system, the components of the binding reactions are the same; however, because of the high ionic strength a lower amount of bulk carrier DNA (2-10-fold lower) is required to abolish nonspecific binding of proteins to the probe than with the low-ionic-strength system.

The protocol is divided into five stages:
(1) Preparation of an end-labeled DNA probe containing a particular protein binding site
(2) Preparation of a low-percentage, high-ionic-strength polyacrylamide gel
(3) Binding of a protein mixture to the DNA probe
(4) Electrophoresis of the binding reactions through the gel
(5) Drying the gel and autoradiography




MATERIALS AND SOLUTIONS

5 mM dNTP Mix (without dCTP) (1 ml)
5 mM dATP ------------------------------------ 50 ul of 100 mM dATP
5 mM dGTP ------------------------------------ 50 ul of 100 mM dGTP
5 mM dTTP ------------------------------------ 50 ul of 100 mM dTTP
Distilled H2O ----------------------------------- 850 ul


DEAE Elution Solution (40 ml)
1 mM Tris-HCl (pH 7.9) ------------------------ 40 ul of 1 M Tris-HCl
1 mM EDTA (pH 8.0) -------------------------- 80 ul of 0.5 M EDTA
1 M NaCl --------------------------------------- 8 ml of 5 M NaCl
Distilled H2O ------------------------------------ 31.88ml
  • Filter through 0.2 um filter.


    5 x Tris-Glycine Stock(pH 8.5) (1 liter)
    Tris base --------------------------------------- 30.28 g
    Glycine ----------------------------------------- 142.7 g
    EDTA ------------------------------------------ 3.92 g
    Add distilled H2O to make a final volume of --- 1 liter


    High-Ionic-Strength Electrophoresis Buffer (4 liters)
    5 x Tris-Glycine Stock -------------------------- 800 ml
    Disitlled H2O ----------------------------------- 3,200 ml


    5 x Binding Buffer (1 ml)
    60% Glycerol ------------------------------------ 600 ul of 100% Glycerol
    60 mM HEPES Buffer (pH 7.9) ----------------- 60 ul of 1 M HEPES Buffer
    20 mM Tris-HCl (pH 7.9) ----------------------- 20 ul of 1 M Tris-HCl
    300 mM KCl ------------------------------------ 300 ul of 1 M KCl
    5 mM EDTA ------------------------------------ 10 ul of 0.5 M EDTA
    5 mM DTT -------------------------------------- 5 ul of 1 M DTT
    Distilled H2O ------------------------------------ 5 ul




    PROCEDURES

    Preparation of an End Labeled-DNA Probe

    1. Gel-purify 1-2 ug of intrerest DNA sequence (25-300 bp), which is digested by restriction endonucleases.
  • Restriction endonucleases should be chosen that generate fragments which are well resolved and that contain at least one 5'-overhang.
  • If 5'-overhangs cannot be generated, the fragment can be labeled using polynucleotide kinase.
  • Klenow-fragment-labeled probes are preferable to kinased probes because some protein extracts contain active phosphatases.
  • Gel-purified, annealed synthetic oligonucleotides may also be used as probes.

    2. Do Klenow reaction as follow:

    10 x Buffer
    1 ul
    Gel-purified DNA (25-300 bp)
    1-2 ug
    [a-32P]dCTP (3,000-6,000 Ci/mmol)
    100 uCi
    5 mM dNTP mix (without dCTP)
    4 ul
    Klenow Fragment
    2.5 U
    Add distilled H2O to make a final volume of
    10 ul


    3. Incubate 20 min at room temperature.

    4. Add 1 ul of 5 mM cold dCTP. Incubate 5 min.

    5. Precipitate the DNA by adding 1/10th vol of 3 M Sodium acetate (pH 7.0) and 2.5 vol of ethanol.

    6. Centrifuge and resuspend the DNA pellet in TE buffer.

    7. Add Gel Loading Buffer to the sample and electrophorese on a agarose minigel (2-4%) containing 5 ug/ml ethidium bromide solution, or native (nondenaturing) polyacrylamide gel (6-8%).

    8. Purify the DNA band by using any gel purification kit available.
  • Alternatively, if gel purification kit is not available, purify the DNA band as follow.

  • Visualize the gel on a UV transilluminator.
  • Using a razor blade, cut a horizontal slit below the band to be recovered.
  • Wet a piece of DEAE membrane in the gel running buffer and slide it into the slit.
  • Squeeze the gel firmly against the paper to close the incision.
  • Insert an extra piece of membrane above the band to keep larger fragments from contaminating it.
  • Alternately, cut away the region of the gel above the band.
  • Resume electrophoresis until the DNA has run onto the membrane.
  • Check on the UV transilluminator.
  • Electrophoresis longer than necessary is not deleterious because the DNA remains on the paper.
  • Rinse the membrane for a few seconds in the electrophoresis running buffer.
  • Blot momentarily on filter paper to remove excess buffer. Do not allow the membrane to dry.
  • Place the membrane in the bottom of a 1.5-ml, round-bottom screw-cap vial containing 400 ul DEAE Elution Solution. Do not crush the DEAE membrane.
  • Incubate 30 min at 68oC.
  • Remove the elution solution from the paper.
  • Place in a 1.5-ml microcentrifuge tube and spin 15 min in a fixed-angle microcentrifuge at 4oC.
  • Transfer the supernatant in a clean microcentrifuge tube, leaving 10 ul at the bottom of the tube undisturbed.
  • Add 4 ul of 1 M MgCl2 to the supernatant. Precipitate with 1.0 ml of 100% ethanol.

    9. Resuspend in 100 ul TE buffer.

    10. Count 1 ul for Cerenkov counts in a scintillation counter to determine cpm/ ul.
  • Efficient labeling should yield at least 5 x 105 cpm/ ul.

    11. Estimate the DNA concentration by ethidium bromide dot quantitation.
  • Efficient recovery of DNA should yield a DNA concentration of 2-10 ug/ ml.
  • Probe can be used for 4-6 weeks.


    Preparation of a Low-Percentage, High-Ionic-Strength Polyacrylamide Gel

    12. Assemble washed 16-cm-long glass plates (siliconized) and 1.5-mm spacers for casting the gel.

    13. Prepare the High-Ionic-Strength Gel Mix as follow.

    5 x Tris-Glycine Stock
    8 ml
    30% Acrylamide:Bis Solution (37.5:1)
    6.3 ml
    50% Glycerol
    2 ml
    Disitilled H2O
    23.7 ml
    Total
    40 ml

  • Filter through 0.2-um filter
  • Store at 4oC for several months.


    14. Mix the following and pour immediately between the gel plates.

    High-Ionic-Strength Gel Mix
    40 ml
    10% Ammonium persulfate
    300 ul
    TEMED
    (N,N,N',N'-tetramethylethylenediamine)
    34 ul


    15. Insert a comb.

    16. Allow the gel to completely polymerize for 20 min.
  • Remove the comb and bottom spacer and attach the plates to the electrophoresis tank after filling the lower reservoir with High-Ionic-Strength Electrophoresis Buffer.
  • Fill the upper reservoir of the tank with High-Ionic-Strength Electrophoresis Buffer.

    17. With a bent-needle syringe, remove air bubbles trapped beneath the gel and flush out the wells.

    18. Pre-run the gel at 100 V for 90 min.


    Binding of a Protein Mixture to the End Labeled-DNA Probe

    19. In a microcentrifuge tube, combine the following:

    5 x Binding Buffer
    2-3 ul
    End-labeled DNA probe
    5,000-20,000 cpm
    (0.1-0.5 ng)
    poly(dI-dC)-poly(dI-dC)
    0.2-1 ug
    BSA
    3-5 ug
    Protein from a crude cell extract
    5-20 ug
    Final volume
    10-15 ul


    20. Mix gently by tapping the bottom of the tube with finger.

    21. Incubate the binding reaction mix 15 min in a 30oC water bath.


    Electrophoresis of the Binding Reactions through the Gel

    22. Load a small volume of 2 x Sample Buffer into one of the wells.
  • Allow the dyes to run into the gel and flush the wells before loading the samples.

    23. Load each binding reaction into the well.
  • Since 5 x Binding Buffer contains glycerol, it is not necessary to add 2 x Sample Buffer to the binding reaction.

    24. Electrophorese at 30-35 mA until the Bromphenol Blue approaches the bottom of the gel (1.5 to 2 hr).
  • Bromphenol Blue migrates at approximately the same position as a 70 bp DNA probe.

  • If electrophoresis is performed at room temperature, the glass plates should be allowed to become only slightly warm.
  • Decrease the voltage if the plates become any hotter.
  • For probes <70 bp, do not run the Bromphenol Blue to the bottom of the gel.
  • To run the gel faster, put the apparatus in a cold room. Higher voltages may then be used without heating the glass plates. In addition, colder temperatures cause a contraction of the gel, increasing its sieving properties. As a result, protein-DNA complexes may appear as sharper bands.

    25. Remove the glass plates from the gel box and carefully remove the side spacers.

    26. Using a spatula, slowly pry the glass plates apart, allowing air to enter between the gel and the glass plate.
  • The gel should remain attached to only one of the plates.

    27. Lay the glass plate (with the gel attached) on the bench with the gel facing up.
  • Place 3 sheets of Whatman filter paper cut to size on top of the gel.

    28. Support both sides with your hands and carefully flip the sandwich over so that the Whatman paper is on the bottom and the glass plate is on the top.

    29. Carefully lift up one end of the glass plate.
  • Peel the Whatman paper with the gel attached to it from the plate.

    30. Cover the gel with plastic wrap and dry under vacuum.

    31. Autoradiograph the dried filter for 2-3 hours exposure with an intensifying screen.




    NOTES




    KIT INFORMATION




    REFERENCES

  • Fried, M, Crothers, DM (1981) Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucl. Acids Res. 9: 6505-6525.

  • Fried, M, Crothers, DM (1984) Kinetics and mechanism in the reaction of gene regulatory proteins with DNA. J. Mol. Biol. 172: 241-262.

  • Fried, M, Crothers, DM (1984) Equilibrium studies of the cyclic AMP receptor protein- DNA interaction. J. Mol. Biol. 172: 263-282.

  • Garner, MM, Revzin, A (1981) A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucl. Acids Res. 9: 3047-3060.

  • Hendrickson, W, Schleif, RF (1984) Regulation of the Escherichia coli L-arabinose operon studied by gel electrophoresis DNA binding assay. J. Mol. Biol. 174: 611-628.

  • Strauss, F, Varshavsky, A (1984) A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell 37: 889-901.

  • Zinkel, SS, Crothers, DM (1987) DNA bend direction by phase sensitive detection. Nature 328: 178-181.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".

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