Protein bound to a small piece of DNA will alter the electrophoretic mobility of that DNA fragment. This allows the analysis of protein-DNA interactions, including the measurement of binding rates, affinity, and specificity. In addition, bound and unbound DNA may be isolated from the gel and used for further types of analysis such as methylation interference or uracil interference.

In the mobility-shift (or gel-shift) assay, end labeled DNA is allowed to bind protein. The resulting DNA protein complexes are then run on a non-denaturing PAGE geland the gel is dried and autoradiographed. The buffer composition of the PAGE gel is varied from the standard TBE gel, because lower ionic strength is needed to facilitate the DNA protein binding. It is interesting to note that binding interactions which can dissociate in free solution within 1 minute show altered mobility on gels which require more than an hour to run. It is theorized that the gel matrix forms a "cage" around the DNA-protein complex, which prevents the components from diffusing away from each other, thus promoting re-association and in effect stabilizing the complex. This would argue that the ratio of bound and unbound material seen on the gel is a direct measure of the fraction of bound material in the sample as it entered the gel. Thus, the conditions set in the binding reaction are of critical importance.

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Mobility Shift Assay

PROBE PREPARATION

A probe of the proper size is cut from 10 µg of plasmid clone, using restriction enzymes which will yield probe of 50-150 bp, with one 5' overhanging end.

Label the probe with ³²P dNTP and Klenow fragment, to fill in the overhang. To the restriction digest reaction, add 100µCi of ³²P dNTP (chosen to be part of the filled in region) and 0.2 mM of the other necessary dNTP's.

Add 3 units of Klenow fragment and incubate 30 minutes at room temperature.

Add an additional aliquot of a mixture of all 4 dNTP's to a final concentration of 0.2mM, and incubate another 10 minutes.

Add 0.1 volume 3M Sodium Acetate, 3 volumes Ethanol, precipitate the DNA and wash once with 70% Ethanol to remove the bulk of the unincorporated label.

Isolate the probe on an agarose gel by electrophoresis onto a DEAE membrane.

Probe should be labeled to 10⁷ - 10⁸ cpm/µg.

BINDING REACTION

Mix the following:

• 10⁴ cpm of DNA probe
• 2µg nonspecific DNA (calf thymus DNA, or synthetic polymer)
• 20 µg sample protein
• 1 µl glycerol in a final volume of 15 µl

Incubate at 30°C for 15-30 minutes.

This gives a basic framework for a binding reaction. Components which may be needed to ensure binding include glycerol (10%), KCl (50mM) and DTT (1mM). Buffer conditions must be optimized for each protein/DNA combination studied.

ELECTROPHORESIS

The mobility shift assay gel uses a low ionic strength buffer system, to avoid salt effects on binding constants. This necessitates buffer circulation between upper and lower chambers to prevent buffer exhaustion and pH shifts during the run.

Prepare 10X Gel Buffer using the following:

• 67mM Tris HCl pH 8.0
• 33mM Sodium Acetate
• 10mM EDTA

Adjust pH of buffer to 8.0.

Use the following buffer at 1X for upper and lower buffer chambers.

Gel Mix:

• 6.7ml 30% acrylamide (AcrylaGel )
• 1.25ml 2% methylene bis-acrylamide (Bis-AcrylaGel)
• 1.25ml glycerol
• 36ml water
• 5 ml 10X gel buffer (made from the previous example)

Just prior to pouring, add 125µl 10% APS and 45µl TEMED to initiate polymerization.

GEL ASSEMBLY

Cast the gel in a standard format (16cm plate) cassette, with 1.5mm spacers and wells at least 0.8cm wide. Pour using standard techniques, and allow to polymerize one hour.

RUN CONDITIONS

Recirculate the buffer at least 10ml/min.

Pre-run gel for 1.5 hours at 6V/cm<.

Preload at least one well with 0.01% Bromophenol Blue in 10% glycerol, to provide a tracking dye. No dyes are included in the binding reactions.

Load binding reactions and run gel at approximately 12 V/cm for 1-2 hours. Adjust the run voltage so that plates do not become warm, because increases in temperature will alter binding equilibria. A current of 25-30 mA is sufficient for a 16 cm gel. Gels may be run at a higher voltage in a cold room.

RESULTS

Bound DNA will appear as a more slowly migrating band, which is not visible in the lanes without protein. The band will disappear with the addition of unlabeled competitive DNA sequences.

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