This method works well for binding conditions in which close to 100 % binding is achieved (assayed by gelshift experiments). The amounts of DNaseI and incubation time may vary.

• prepare DNA-fragment (100 to 300 bp in length) by cutting it with one 5' overhang-producing restriction enzyme and one 3' overhang-producing restriction enzyme
• label DNA-fragment at the 3' end using a Klenow-DNA Pol. fill-up reaction or a SuperScript RT reaction
• setup binding reaction in 20 µl total volume:
  10000 - 20000 cpm labeled fragment, 4 µl 5 x DNaseI binding buffer, protein, H₂O
• incubate 10 min at room temperature
• dilute DNaseI ( i.e. RNase free DNaseI 10 U/µl; Roche Diagnostics)
  with DNaseI dilution buffer to 0.5 - 1 U/µl
• add 1 to 3 U DNaseI to each binding reaction
• incubate 60 to 120 sec. at room temperature
• stop reaction by adding 100 µl phenol, vortex immediately
• add 80 µl H₂O, 100 µl chloroform, vortex
• spin for 2 min in tabletop centrifuge
• disgard supernatant, add 3 µl glycogen (2 µg/µl), 15 µl 3 M NaOAc, 600 µl Ethanol
• incubate at least 30 min. at -80 °C
• spin 10 min. in tabletop centrifuge, wash with 70 % Ethanol, dry pellet
• resuspend pellet in 3.5 µl sequencing stop solution
• heat sample 3 min. at 95 °C, chill on ice
• load onto 8 % sequencing gel
• as marker we use pBR322 MspI cut, labeled with T4 Polynucleotide kinase

 5 x DNaseI binding buffer:

100 mM Tris pH 7.5; 500 mM NaCl; 25 mM MgCl₂; 5 mM EDTA; 25 % glycerol; 0.1 µg/µl poly[dIdC].

 DNaseI dilution buffer:
2 mM MgCl₂; 2 mM CaCl₂; 20 mM Tris pH 7.5; 25 % glycerol.

 sequencing stop solution:
95 % Formamide; 20 mM EDTA; 0.05 % Bromphenol blue and Xylene cyanole FF each.