The overall goal of this assay is to detect protein-DNA interactions using non-radioactive probes. This method can help answer key questions in the molecular biology and biochemistry fields, such as determining DNA target sequence of proteins. The main advantage of this technique is that it is an easy, safe, and time saving alternative to using radioactive probes.
To begin this procedure, prepare a five percent native polyacrylamide gel containing 0.5x tris borate EDTA or TBE, and 2.5%glycerol using a mini protein gel system. To prepare 30 milliliters of gel solution for four gels, mix distilled water, TBE, ammonium persulfate, TEMED, acrylamide bis, and glycerol. Pass the gel solution immediately.
After polymerization, wrap the gels in clear plastic wrap pre-wetted with 0.5x TBE and store them at four degrees Celsius. Next, design a long oligonucleotide for approximately 51 mers. Design complementary short oligonucleotides for approximately 14 mers with infrared fluorescent dye modification at the five prime terminus.
Once the oligonucleotides have been prepared, resuspend them in 1x Tris-EDTA buffer to a final concentration of 100 micromolar. For annealing, mix 0.6 microliters of five prime dye short oligonucleotide, 1.2 microliters of long oligonucleotide, and 28.2 microliters of sodium chloride Tris-EDTA buffer in a 1.5 milliliter tube. Place the tube in boiling water for five minutes, then turn off the heat source and allow the water with the annealed oligonucleotides to cool overnight in the dark.
To make double-stranded DNA probes, mix 30 microliters of the annealed oligonucleotides with DNTPs, Klenow buffer, Klenow five prime dye tag, and double distilled water in a 0.2 milliliter PCR tube. Incubate the mixture for 60 minutes at 37 degrees Celsius in a PCR machine. Then, add 3.4 microliters of 0.5 molar EDTA to stop the reaction, and heat inactivate at 75 degrees Celsius for 20 minutes in the PCR machine.
Following this, dilute the filled in probes with sodium chloride Tris-EDTA buffer to a final concentration of 0.1 micromolar. Store the oligonucleotides at minus 20 degrees Celsius in the dark until ready to use. To prepare unlabeled probes, mix 20 microliters of long oligonucleotide, 20 microliters of Long R oligonucleotide, and 60 microliters of sodium chloride Tris-EDTA buffer in a 1.5 milliliter tube.
Place the tube in boiling water for five minutes, then turn off the heat source and allow the water with the annealed oligos to cool overnight. Prepare one 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 one hour, or until the current no longer varies with time.
Next, mix four microliters of 5x binding buffer, 80 to 200 nanograms of purified protein A, one 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 millimeters for focus offset. Select the area that the gel occupies on the scanner.
Finally, click Start to begin the scan. The progress of electrophoresis can be visualized with Orange G loading dye, whereas Bromophenol blue is detected during scanning and therefore interferes with image analysis. Addition of dI-dC to the binding reaction abolished the binding of purified 6xHis-SOX-2 with the five prime dye DNA probes.
The LIM-4 mutant and SOX-2 cold probe mutated in the LIM-4 binding site is as efficient as the wild type cold probe in competing with the infrared fluorescent dye labeled probe for binding to 6xHis-SOX-2. However, the competition efficiency of the LIM-4 and SOX-2 mutant cold probe mutated in the SOX-2 binding site is much lower than the wild type cold probe for binding to 6xHis-SOX-2. Supershift assays of the SOX-2 DNA complex using 6xHis and flag epitopes resulted in a supershifted SOX-2 DNA band.
Once mastered, this technique can be done in two to four hours if it is performed properly. While attempting this procedure, it is important to remember to keep the infrared probes in the dark. Following this procedure, other methods like CRISPR cas9 genome editing can be performed in order to answer additional questions like the importance of a particular DNA binding sequence.
After its development, this technique paved the way for researchers in the field of biochemistry to explore protein-DNA interactions in various model organisms. After watching this video, you should have a good understanding of how to determine protein-DNA interactions using non-radioactive DNA labels. Don't forget that working with acrylamide can be extremely hazardous and precautions such as personal protective equipment should always be taken while performing this procedure.
