Amersham typhoon rgb imager

The preparation of samples for electrophoresis was performed using the protocols published earlier [35 (link),44 (link)]. Briefly, for the electrophoresis aimed to study the integrity of the EGCG–nucleosome complexes, EGCG (6–100 µM) was added to nucleosomes (60 nM) in 0.01 M buffer for dialysis (10 mM NaCl, 10 mM Tris-HCl (pH 7.9), 0.1% NP40, 0.2 mM EDTA, 5 mM β-ME), incubated for 20 min, mixed with sucrose (final concentration 10%) and loaded into the 4% polyacrylamide gel. Electrophoresis was performed in the HE buffer (10 mM HEPES-NaOH pH 8.0, 0.2 mM EDTA) at +4 °C at 80 V.
In the PARP1-related studies, nucleosomes (2–3 nM) were preincubated with EGCG (0.8–10 µM, 15 min, 25 °C) and further incubated with PARP1 (20 or 50 nM) or with PARP1 and NAD+ (20 μM) in the TB150 buffer (45 min, 25 °C). The probes were subjected to electrophoresis in the 4% polyacrylamide gel (acrylamide: bisacrylamide 59:1; 0.2× TBE buffer) at 120 V for 90 min. Ladder GeneRuler 100 bp (Thermo Fisher Scientific, Waltham, MA, USA) was used as a marker.
The fluorescent imaging of the gels was performed using the Amersham Typhoon RGB imager (GE Healthcare Bio Sciences AB, Uppsala, Sweden).

Radiolabelled intron precursor RNA (and mutants) were mixed with purified maturase protein (and mutants) under near-physiological condition (50 mM K-HEPES pH 7.5, 150 mM KCl and 5 mM MgCl₂) to perform the in vitro forward splicing assay. To do so, radiolabelled intron RNA was first mixed with buffer and water and heated to 95 °C for 1 min, after which it was returned to 37 °C for 5 min. Potassium chloride and maturase protein stocks were added and the sample was incubated at 37 °C for another 5 min. Magnesium chloride stock was subsequently added to the mixture to initiate the splicing reaction. The final concentration of radiolabelled intron precursor RNA was 5 nM and maturase protein was 20 nM. After incubation at 37 °C for 1 h, 2 μl of the reaction mixture was taken out and quenched by mixing with an equal volume of 2× formamide loading dye (72% (v/v) formamide, 10% sucrose, 0.2% bromophenol blue dye, 0.2% xylene cyanol dye and 50 mM EDTA) precooled on ice. Samples were analysed on a 5% urea denaturing polyacrylamide gel. The gel was dried and used to expose the phosphor storage screen overnight. The screen was then imaged on an Amersham Typhoon RGB imager (Cytiva). The bands were quantified using ImageQuant TL 8.2 (Cytiva).

All reactions were carried out in a buffer containing 20 mM Hepes (pH 8.0) at 37 °C, 100 mM NaCl, 0.1 mg/ml bovine serum albumin, and 1 mM DTT. All components were assembled on ice. For each reaction, the reaction mixture was pre-equilibrated for 5 min on ice and then equilibrated for 2 min at 37 °C before initiating the reaction with 5 mM MgCl₂. All listed concentrations are the final concentrations. At specific time points, an aliquot of the reaction was removed and quenched with a solution containing 95% (v/v) formamide, 50 mM EDTA, 0.025% (w/v) bromophenol blue, and 0.025% (w/v) xylene cyanol. Samples were analyzed on a 16% denaturing PAGE gel containing 7 M urea. Gels were imaged on an Amersham Typhoon RGB imager (Cytiva) and quantified using the ImageQuant software (Cytiva). The data were analyzed using GraphPad Prism (version 6.0; GraphPad Software, Inc). Initial velocities at varying concentrations were obtained by fitting the product intensities to linear regression. The obtained k_obs were fit to the Michaelis–Menten equation to obtain k_cat and K_M.