Radio labeled γ 32p atp

Radio-labeled [γ-32P] ATP (6000 Ci/mmol, 150 mCi/mL) was purchased from Perkin Elmer. Restriction endonucleases, T4 DNA ligase, T4 polynucleotide kinase (PNK), and T7 RNA polymerase were purchased from New England Biolabs and used according to manufacturer’s instructions. RNase-free DNase I (Roche), AMV reverse transcriptase (Life Sciences), and Pfu DNA polymerase (Stratagene) were used according to the manufacturer’s specifications. DNA oligonucleotides were purchased commercially (IDT). The lacZ-specific oligonucleotide 5’-GTTTTCCCAGTCACGACGTTG-3’, which anneals to positions +99 to +78 of the lacZ coding sequence in lacZ fusions, was used in the primer extension inhibition (toeprint) assays.

Samples were prepared for AFM as described above. After rinsing, 100 μL 3 mM radiolabeled [γ-³²P] ATP (0.908 nCi/μL, PerkinElmer) in imaging buffer was added to the surface. At set time points, 4 μL of the surface solution was sampled and mixed with 4 µL of 50 mM ethylenediaminetetraacetic acid (EDTA). The resulting solutions for each timepoint were spotted onto a thin-layer chromatography (TLC) plate (Millipore Corporation) and allowed to dry. Separation of species was performed by submerging the bottom of the TLC plate in 125 mM KH₂PO₄ solution for 1 h. Then the plate was dried and exposed to a phosphor imaging plate (Fujifilm BAS-MS 2340) for 15 min in a closed cassette. The imaging plate was scanned in a phosphor imager (FLA3000) and analyzed using ImageQuant software. The percent ATP hydrolyzed was quantified as the relative amount of radioactive inorganic phosphate species that migrated from the ATP spot.