Model 583 gel drier

Oligonucleotides (Invitrogen) at a concentration of 100 μM were annealed to form G4s by heating at 99°C for 20 min and slowly cooling to 4°C over several hours in 10 mM Tris-HCl (8.0) and 100 mM KCl. Oligonucleotides analyzed by polyacrylamide gel electrophoresis (PAGE) were radiolabeled following standard protocols using T4 polynucleotide kinase and [γ-³²P] ATP. Oligos were then purified using ProbeQuant G-50 microspin columns (GE). DHX36 was incubated for 30 min at 37°C with 10 pM radiolabeled, annealed Z33 G4 in K-Res buffer supplemented with 10 mM EDTA in a volume of 50 μl. Following incubation, the reaction was directly loaded onto a 6% polyacrylamide gel (37.5:1) (National Diagnostics) and run at 30 mA for 1.5 h. When binding inhibition was studied through the use of a ligand, ligand was added at indicated concentrations after the initial 30 min incubation. Experiments with ligand titration contained 4 nM DHX36 and 4 nM Z33 G4. The mixture was then incubated for an additional 10 min at 37°C before being analyzed by PAGE. Gels were either directly imaged or dried using a BioRad Model 583 Gel Drier and exposed to a storage phosphor screen followed by visualization with a Typhoon Trio scanner (GE). Bands were quantified using ImageQuant TL software. All ligand binding data were fit with three-phase exponential decay functions.