ELECTROPHORETIC MOBILITY SHIFT ASSAY (EMSA) OF PROTEIN-DNA INTERACTIONS

Introduction

          The electrophoretic mobility shift assay (EMSA) of DNA-protein interactions is a simple and quick, and sensitive method for the detection of sequence-specific DNA binding proteins in complex extracts. It can also be used to study the binding activities (complex formation) of purified or recombinant sequence-specific DNA-binding proteins, or the binding activities of proteins that bind DNA in a non-sequence-specific manner (e.g. histones). Additionally, it can be used to determine the affinity, abundance, association and dissociation rates and binding specificity of DNA binding proteins. The basis of the EMSA assay is that protein-DNA complexes are surprisingly stable to electrophoretic fractionation in gels and migrate as distinct bands more slowly than the free DNA fragment.  Thus, the appearance of one or more slowly migrating bands may be taken to indicate that one or more proteins present in the extract bind in a sequence-specific manner to the binding-site DNA fragment. 

          The fractionation system most frequently used is non-denaturing polyacrylamide gels, but agarose gels can also be used.  The size of the DNA-protein complex (or complexes) may vary considerably, and is often unknown at the beginning of the study. 

General considerations

          When studying sequence specific protein-DNA interactions it is helpful to keep in mind a few simple facts.

i)  The binding constants of sequence-specific DNA binding proteins vary over a wide range (generally between 10-9 and 10-14 M).  Consequently the molarity of protein or binding-site DNA required for complex formation is dependent on the binding constant and is likely to be different for different DNA-binding proteins.

ii)  The concentrations of different DNA-binding proteins in a cell extract might be both variable and low.  Therefore, when setting up a preliminary binding experiment, it is advisable to use a relatively high concentration of binding-site DNA (10-9-10-8 M) to facilitate protein-DNA complex formation, even if protein is limiting.

iii)  Cell extracts contain many DNA-binding proteins that are capable of interacting with DNA in a sequence-independent manner and thus they may compete with the protein of interest for binding to a specific DNA.  This problem can be minimized by the inclusion of competitor (or carrier) DNA in the binding reactions.  We normally use either sonicated salmon sperm DNA (to an average size of 500 bp), or synthetic polynucleotides e.g. poly (dI-dC)-poly (dI-dC) [Pharmacia].  Some people use E. coli DNA or synthetic poly (dA-dT)-poly (dA-dT) as competitors.  The quantities of nonspecific competitor included in a binding reaction usually vary from 0.5 mg to 2 mg when crude extracts are analyzed (2-20 mg of nuclear extract protein).

iv)  When attempting to define a DNA-binding activity or target DNA, consider the possibility that one protein might bind specifically to more than one structurally related sequences and, conversely that a sequence may be recognized by more than one protein.

v)   The conditions required for optimal complex formation and stability for different proteins may vary considerably in terms of pH, ionic strength and metal ion content.  In this respect you should consider the conditions given below (in the “Technical aspects of EMSA” section) just as an example, or case report, rather than as the ten commandments of the Holy Bible.

vi)  Differences in the size, aggregation state and pI of protein-DNA complexes affect the choice of conditions used in the EMSA assay.  Consider that  the process of electrophoretic separation might destabilize protein-DNA complexes.  The ionic strength and composition of gel loading and electrophoresis buffers should therefore be adjusted in accordance to the known or suspected properties of the particular protein-DNA complex.

Technical aspects of EMSA

Binding reactions.  Combine the following in an Eppendorf tube (be especially careful to avoid generating air bubbles when mixing).

          4 ml    5X binding buffer (see below)

          2 ml    BSA (3 mg/ml)

          1 ml    nonspecific competitor DNA (e.g. poly[dI-dC], 2 mg/ml)

          2 ml    nuclear or total extract (2-20 mg protein)

          9 ml    H2O

          Mix carefully by swirling the pipette tip, while avoiding generating air bubbles, and incubate at room temperature for 5-10 min.

Then, add 2 ml DNA probe (10-30,000 cpm, usually 0.1-0.5 ng).  The final volume of the reaction is 20 ml.  Incubate at room temperature for 30 min.

Load the reaction mixtures directly on the native gel, that has been pre-run for 30-45 min at 120 Volts.

 

5X stock binding buffer

          50 mM Tris-HCl pH 7.5

          250 mM NaCl

          25% glycerol

          5 mM EDTA

          20 mM DTT

 

Gel preparation:   I normally use native 4% gels in 0.25X TBE buffer.  The gel is prepared by combining the following reagents:

          6.5 ml Acrylagel

          3.3 ml Bis-acrylagel

          2.5 ml 5X TBE

          5 ml             25% glycerol

          32.5 ml        H2O

Mix by swirling gently and then add:

          50 ml           TEMED and

          135 ml          30% (w/v) ammonium persulfate.

 

**NOTE:  Acrylagel (National Diagnostics) is a stabilized 30% acrylamide solution and Bis-acrylagel is a 2% bis-acrylamide solution.  You can always use your home-made acrylamide and bis-acrylamide solutions as long as they are of very good quality (Remember you are running a native gel and the well-being of your protein is very important).

 

Normally, I use 20 X 20 cm gels (1.5 mM thick) and the amount given above (50 ml) can prepare one gel.  Pour the gel carefully avoiding air-bubbles.  Then insert the comb.  The gel plates, spacers and comb should be very clean and special care should be taken to remove traces of detergent that may be left overs from either the wash of these components or from the person who run an SDS-PAGE gel in the same apparatus before you (Remember you ARE running a native gel).

          To generate perfect wells I wash the comb with clean paper tissue that has been soaked in the aforementioned acrylamide solution.  This takes care of possible oxidizing agents (traces of detergents) that could inhibit polymerization around the wells.

          Prepare, your gel several hours ahead (or the previous day and store in the cold-room covered with Saran-Wrap) to allow complete polymerization.  Do not remove the comb before you are ready to run the gel, otherwise your wells will dry out, if not covered with buffer.

 

Electrophoretic separation:  This is one of the most critical steps and particular care should be taken to avoid sloppy loading practices, overheating and pH changes due to buffer exhaustion.

          The comb is carefully removed (one end first then the other) and the wells are rinsed immediately with distilled water.  The gel is assembled in the electrophoresis unit, electrophoresis tank buffer is added and the wells are rinsed with electrophoresis tank buffer using a Pasteur pipette.  Well imperfections can also be corrected at this stage using a long needle.

          The electrophoresis tank buffer is 0.25X TBE.  If the gel is to run at room temperature no previous preparation is required, but if you are to run the gel at 4˚ C (cold room) then both the gel and the electrophoresis tank buffer should be pre-equilibrated at this temperature.

          The gel is pre-run for 30-45 min at 120 Volts.  The buffer is re-circulated during the run to prevent buffer exhaustion, which will result in pH changes in the buffer and within the gel.

          Just before you load your samples rinse the wells with tank buffer once again.  I usually load my samples while running the gel at 100 V because this condition should drive the complexes faster into the gel and reduce the possibility of mixing of the sample with the tank buffer during loading.

          The samples should not contain any loading dyes to avoid interference with binding.  The only way to visualize them, while loading, is to look for the differential refraction of the denser sample (due to the presence of glycerol) compared to that of the tank buffer. In general it is quite difficult to monitor the quality of loading. I recommend the use of the kind of pipette tips used for sequencing to avoid mixing with the tank buffer.  You should also avoid the generation of air-bubbles during loading that could also lead to sample mixing with the tank buffer.

          One lane is usually loaded with sample buffer containing dyes (0.1% xylene cyanol and 0.1% bromophenol blue) to monitor the progress of the electrophoresis.  When using 4% gels, the bromophenol blue runs with 60 bp DNA fragments and the xylene cyanol with 200 bp DNA fragments.

          After loading, increase the voltage to 160-180 Volts and run the gel till the bromophenol blue is close to the bottom of the gel.

         

Autoradiography:  After the end of the electrophoresis pry the gel plates open with a spatula.  The gel will remain stuck on one plate.  Put two precut 3MM Whatman papers on top of the gel, then a glass plate on top of them and invert the sandwich.  Now the papers are at the bottom and the gel at the top.  Remove the upper glass plate and the gel should remain attached to the Whatman paper.  Cover with Saran wrap and dry at 80˚ C under vacuum for one hour.  After the gel is dried you can expose it to an X-ray film with an intensifying screen at -70˚ C, or without at room temperature.