Electrophoretic Mobility Shift Assay for Detection of Transcription Factor-DNA Complexes

Regulatory proteins, like transcription factors, recognize and bind to specific DNA sequences in promoter regions, regulating gene expression. 

To detect specific transcription factor-DNA interactions using the electrophoretic mobility shift assay, begin with a tube containing a desired transcription factor and specific short, infrared, fluorescent dye-labeled DNA probes in a binding buffer. Incubate in the dark, preventing dye photobleaching.

The buffer's ionic strength and pH provide suitable conditions for the transcription factor to identify and bind specific binding sequences on the DNA probe. Magnesium ions in the buffer further stabilize the protein-DNA complexes.

Add a suitable loading dye to the mixture, helping visualize electrophoresis progression. Load this mixture and DNA probes into the wells of a precast, non-denaturing polyacrylamide gel.

Perform electrophoresis in the dark.

The applied electric field causes low molecular weight, unbound, negatively-charged DNA probes to move through the gel toward positively-charged anodes. However, the large protein-DNA probe complexes — with a higher molecular weight — migrate slowly, leading to a delayed migration and shift in the mobility of DNA in the gel.

Post-electrophoresis, scan the gel using an infrared imaging system, visualizing the infrared fluorescent dye-labeled DNA probes.

A free DNA probe band is at the bottom of the gel, while protein-DNA complexes appear as a shifted band on the gel, indicating protein-DNA complex formation.

Prepare 1 milliliter of 5X binding buffer by mixing Tris-HCl, sodium chloride, potassium chloride, magnesium chloride, EDTA, DTT, BSA, and double-distilled water.

Prior to setting up the binding reactions, pre-run the 5% native polyacrylamide gel in 0.5X TBE and 2.5% glycerol to remove all traces of ammonium persulfate at 80 volts for 30 minutes to 1 hour or until the current no longer varies with time.

Next, mix 4 microliters of 5X binding buffer, 80 to 200 nanograms of purified protein A, 1 microliter of 0.1-micromolar dye-conjugated probe, and double-distilled water. Incubate the mixture at room temperature in the dark for 15 minutes.

Following incubation, load all of the binding reaction onto the gel, and run the gel at 10 volts per centimeter to the desired distance. Cover the gel apparatus with aluminum to keep the gel in the dark as much as possible.

Clean the scanner bed of an infrared imaging system with distilled water. Wipe-dry the glass plates containing the gel, and place them on the scanner bed. Open the infrared imaging software, and click on the "Acquire" tab.

For thicker plates, use the settings of '700' for "Channel," 'Auto' for "Intensity," '84 micrometers' for "Resolution," 'Medium' for "Quality," and '3.5 millimeter' for "Focus Offset." Select the area that the gel occupies on the scanner. Finally, click "Start" to begin the scan.