The overall goal of this procedure is to demonstrate a quantitative DNA binding assay, which is useful in examining protein DNA interactions, discovering transcriptional regulators of gene expression and in identifying novel anti-tumor agents. This is accomplished by first preparing a streptavidin coated assay plate with an appropriate BIOTINYLATED DNA target. The second step is to add nuclear extract proteins with or without potential inhibitors and allow them to bind with the DNA target.
Next, the bound transcription factors are detected with primary antibodies and horse radish peroxidase coupled secondary antibodies. The final step is to add a horse radish peroxidase substrate to the wells in order to elicit color development. Ultimately, color development is measured with the spectrophotometer.
Over time, the amount of color change will indicate the degree of DNA binding and the effect of potential inhibitors of DNA binding. The main advantage of this technique over other techniques, such as electrophoretic mobility shift assay, is that it is a quantitative DNA inhibitor binding assay. To begin, prepare an adequate number of cell cultures consisting of at least three times 10 to the seven cells for each condition tested.
Here, human bone marrow endothelial cells are being used, which express the run X two protein. Once the cells are 80%confluent. Treat the cells with 0.2 micrograms per milliliter, no conazole to arrest cell growth at the G two M cell cycle boundary.
This will stabilize the run X two protein and produce maximal DNA binding after 16 hours. Lyce the cells and isolate the protein as directed in the accompanying text protocol. Once the protein has been isolated, measure the protein concentration using a standard BCA assay.
Then store the samples at minus 80 degrees Celsius in 10 microliter aliquots with concentrations between two and five milligrams per milliliter. Fix a 96 well plate with a 0.1 molar solution of sodium carbonate by adding 300 microliters of the mixture per. Well Prepare enough well so that each sample is run in triplicate.
Incubate the plate for two hours on a rocking platform at room temperature. Then wash the plate three times with 300 microliters of strep avid and wash buffer and incubate with 100 microliters of biotin labeled double stranded oligonucleotides at 1.25 nanomoles per well for two hours while rocking. Next, wash the plates three additional times with the strep din wash buffer.
Then add 100 microliters per well of the master mix containing one XDNA binding buffer DNA binding proteins at three to nine micrograms per well and poly DIGC at one microgram per well. Then add the master mix containing any potential inhibitor such as vitamin D three or the appropriate dilution of the solvent control, such as ethanol to their respective wells. Then cover the plate and place it on a rocking platform at four degrees Celsius overnight night following incubation with the nuclear extracts.
Wash the plate three times with the strep avid and wash buffer. Then add 90 microliters per well of a run X two specific monoclonal antibody diluted to 0.2 nanograms per microliter and strep avid and wash buffer. Cover the plate and incubate it at room temperature on a rocking platform for an hour.
Then rinse the wells again with strep avid and wash buffer and add 90 microliters per well of the secondary antibody mixture containing five nanograms per microliter fab specific affinity purified HRP conjugated antibody diluted in the strep. AVID and wash buffer. Incubate the secondary antibody for 30 minutes at room temperature on a rocking platform.
Then wash it six times with 300 microliters per well of Strp and wash buffer. Next, develop the results by adding 50 microliters of a tetraethyl benadine substrate to each well directly from the stock bottle for continuous monitoring of the samples. Place the samples into the spectrophotometer and measure the absorbance at 635 nanometers continuously.
For a one-time measurement, incubate the samples for 10 to 20 minutes at room temperature in the dark. During this time, the wells should change from clear to varying degrees of blue. Stop the reaction by adding 50 microliters of sulfuric acid to each.
Well then measure the absorbance of each well at 450 nanometers using a 96 well plate reader. The graph shown here is an example of continuous monitoring of the effect of biologically active vitamin D three on run X 2D NA binding. The concentrations tested had little effect on run X 2D NA binding.
Although other vitamin D three compounds have been shown to have dramatic effects. The compound 5 2 2 1 9 7 5 tested here was identified using computer-assisted drug design. Upon testing, the compound exhibited a dose-dependent inhibition of rux two binding from one nanomolar to 100 micromolar 50%of the renx 2D NA binding was able to be inhibited with just 0.01 micromolar of the compound.
After watching this video, you should have a good idea of how to perform the DNA binding assay and test potential inhibitors of DNA binding.
