Preparation of DNA substrate:

DNA to be analyzed must be cloned in such a way as to present a restriction site for an enzyme leaving a 5' overhang (3' recessed OH) 50-150 bp from the putative binding site. This site is labeled by "filling in" the recessed site with 32P-dNTP's using DNA polymerase. The probe is then cut from the remaining plasmid by a second restriction enzyme, 150 bases on the opposite side of the binding site. This releases a 200-300 bp probe, labeled at one end (See figure below).

Digest 5-10 picomoles of plasmid (10-20µg of a 3000bp construct) with an enzyme which will leave a recessed 3' end 50-150bp from the binding site.

Ethanol precipitate the DNA, and wash 1X with 70% Ethanol.

Add 50 µl of 1X Klenow buffer containing 50 µCi of each 32P dNTP. Add 10 units of Klenow fragment in < 2 µl, and incubate 30 minutes at 25°C.

Chase reaction with 5 µl of 2mM each dCTP, dGTP, dTTP, dATP, to ensure complete polymerization.

Ethanol precipitate twice with 0.1 vol 3M Sodium Acetate and 3 volumes of Ethanol, or purify with a spin column or glass powder elution to remove unincorporated label.

Cut with a restriction enzyme to release a 200-300 bp end labeled probe.

Run probe on a 1.5-2% agarose gel and recover fragment by electroelution. Further purification may be necessary to ensure consistent results. Ion exchange mini columns provide good results.

If volume is >50µl, ethanol precipitate the probe, and reconstitute in 50 µl TE buffer.

Bind Protein to DNA Probe:

Mix 105 cpm of probe with 200 µl of DNase Footprinting Buffer:

10mM Tris HCl, pH 7.6 4mM MgCl2 1mM CaCl2 150mM KCl 2mM DTT 100 µg/ml BSA 2mg/ml calf thymus DNA pH 7.6 (adjust buffer if necessary)

Add 20 µl protein sample. A series of dilutions covering 4-5 orders of magnitude will allow calculation of the binding affinity.

Prepare a blank tube with 20 µl of Assay buffer.

Incubate 30-45 minutes, at equilibration temperature.

The optimum time and temperature must be determined for each DNA/probe combination.

DNase I Treatment:

DNase treatment proceeds for only 2 minutes, so stop solution and a dry ice Ethanol bath must be prepared before beginning the treatment.

Cool Stop Solution to -70°C prior to use.

Prepare DNase I solution:

The amount of DNase I required will vary depending upon the purity, age and storage conditions used for the enzyme. Start with 0.1 mg/ml DNase I and adjust to get an average of 1 nick per DNA molecule.

Dissolve DNase I in Assay/Equilibration buffer without BSA or calf thymus DNA.

To each 200 µl sample of protein/DNA, add 5 µl DNase I solution.

Reproducible pipetting is essential at this stage if different DNAµprotein ratios are to be compared.

Incubate at Equilibration temperature exactly 2 minutes, then add 800 µl Stop Solution @-70°C.

Precipitate DNA at -70°C for 30 minutes. Wash pellet with 70% Ethanol twice, and remove all supernatant. Air dry or speedvac 10 minutes.

Redissolve pellet in 6 µl of loading buffer, and run on a standard denaturing PAGE gel.