Molecular imager quantity one software

Crosslinking of PARP-1 was carried out in reaction mixtures (20 μl) that contained 40 mM Tris-HCl (pH 7.5), 40 mM NaCl, 10 mM MgCl₂, 0.1 μM (DNA*pF or DNA*p) and 0.1 μM human recombinant PARP-1 or BTNE (1.25 mg/ml). To these mixtures, 0.5 mM NAD⁺, 1.2 μM p21 or p21Cter were added, as indicated in the figure legends. The reaction mixtures were incubated at 37°C for 3 min then irradiated by UV light (λ = 312 nm, 1.5 J/cm²) on ice. A Bio-Link BLX-312 cross-linker (Vilber-Lourmat) was used as light source in all experiments. Reactions were stopped by adding SDS-sample buffer and heating for 5 min at 96°C. Photocrosslinking products were separated in a 10% SDS-PAGE. The gels were dried and subjected to phosphor imaging for quantification using Molecular Imager/Quantity One software (Bio-Rad, USA) or “Typhoon” (Amersham Pharmacia Biotech, USA).

The reaction mixtures (20 μl) contained 20 mM Hepes, pH 7.5, 100 mM NaCl, 5 mM MgCl₂, 0.1 mg/ml BSA, 200 nM 5′-[³²P]-labelled AP site-containing DNA (DNA-AP) and 0.15 mg/ml protein extracts. AP sites were generated immediately before experiments by treatment of the DNA duplex with E. coli uracil DNA glycosylase (5 U/pmol of the DNA duplex). The reaction mixtures were incubated at 37 °C for 30, 60 and 90 s. The AP endonuclease reactions were stopped by adding of 90% formamide, 10 mM EDTA, 0.1% bromophenol blue, and 0.1% xylene cyanol. The mixtures were heated at 90 °C for 3 min, and the products were separated by electrophoresis in 20% polyacrylamide gel (PAGE) containing 7 M urea. The gels were dried and subjected to phosphorimaging for quantification using Molecular Imager/Quantity One software (Bio-Rad, USA). Processing of AP site resulted in the appearance of ~15 nt band.