1. To a sterile 1.5-ml microfuge tube add: 32P-labeled target DNA 1 ng (1-10 fmoles) 1 mg/ml poly(dI-dC) 1 pl nuclear extract (5-10 |jg) < 10 pl or protein fraction <10pl
20% Ficoll 400 5 pl or
10% polyvinyl alcohol 4 pl H2O to 20 pl
Include control reactions with every experiment. Positive control reactions contain a nuclear extract (or protein fraction) and a radiolabeled DNA fragment carrying a sequence recognized by a DNA-binding protein that is abundant in the extract and has high affinity for the DNA sequence. Examples are a DNA fragment containing an Sp1, C/EBP, or NF-1 site and mammalian cell nuclear extract, or a lacI recognition site and extract derived from a lacIq strain of E. coli. The negative control reactions contain the radiolabeled target DNA fragment, but no nuclear extract.
2. Centrifuge the reaction tubes for several seconds in a microfuge to deposit the reaction mixtures at the bottom of the tubes. Incubate the reactions for 10-30 minutes on ice.
3. Add 3 pl of 6x gel-loading buffer I to each tube. Load the samples into the slots of a neutral 4-7% polyacrylamide gel.
4. Run the gel in either 0.5x Tris-glycine buffer or 0.5x TBE buffer at 200-250 V and 20 mA for > 2 hours. Depending on the lability of the binding protein(s) and the affinity of the binding reaction(s), it may be necessary to run the gel at 4°C.
5. After electrophoresis is complete, pry the gel plates apart, transfer the gel to a piece of sturdy blotting paper, and dry the gel for approx. 1 hour on a gel dryer.
6. Expose the dried gel to X-ray film for > 1 hour at -20°C to visualize radiolabeled DNA fragments. Less abundant DNA-protein complexes can be detected after 1-3-hours on a phosphorimager.
Was this article helpful?