Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

To prepare the titration and calibration wells of a 96-well plate, transfer two gel stock aliquots to a 75-degree Celsius incubator shaker.

For each titration well, add 1.4 nanomoles reference DNA, transcription factor protein at a final concentration of 20 to 60 nanomoles, 0.2 millimolar DTT, and the binding buffer to 240 microliters of the melted gel.

Mix thoroughly by inverting and shaking the tube. Then, slowly pipette 200 microliters per well of the DNA-containing gel in the designated titration wells of a 96-well plate. For each calibration well, add 5 nanomoles Nile blue dye to 240 microliters of the melted gel. Avoiding air bubbles, slowly pipette 200 microliters of the dye-containing gel solution in five to six wells of the 96-well plate.

Place the gel on a perfectly horizontal surface and incubate for 10 minutes at room temperature, and another 10 minutes at 4 degrees Celsius. Use a multiwell plate reader to check the homogeneity of the gel height levels in different wells of the plate.

To prepare the competitor DNA solution, first, combine the labeled reference DNA, the protein, and the three-fold concentrated binding buffer. Then, mix 20 microliters of the solution with 40 microliters of each of the annealed competitor DNA solutions.

For each calibration well, combine the appropriate amounts of one of the annealed competitor DNA solutions and the three-fold concentrated binding buffer containing 15-millimolar Nile blue dye solution. Finally, add 50 microliters of the mixed competitor solutions on top of the gels as simultaneously as possible.

To begin image acquisition, place the 96-well plate on the microscope stage. Then, take time series of z-stack images of wells until complete unbinding of the protein from the reference DNA.

Perform the titration assay according to the manuscript. Then, use HiP-FA software to create titration curves for individual competitor sequences. Then, click the "Export" button to obtain the dissociation constant and concentration of the active protein in each titration well.