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