Ge illustra probequant g 50 micro column

Nuclear extracts containing K-RTA and K8 from HEK293 cells were prepared by transiently transfecting K-RTA and K8 expression plasmids. Binding of these proteins to DNA were evaluated by EMSA using ³²P-labeled probes containing specific K-RTA or K8 binding motifs sequences. The probes were synthesized as double-stranded DNA containing three copies of motif sequences in tandem (RV: 5’-AGAAAAATTCAGAAAAATTCAGAAAAATTC-3’, RB: 5’-AGAGACAGGAAGAGACAGGAAGAGACAGGA-3’, KB: 5’-AAAAATAAAAAAAAATAAAAAAAAATAAAA-3’). These probes were end-labeled with α-³²P and purified on a GE Illustra ProbeQuant G-50 micro column (GE Healthcare, USA). A 50 μl DNA-protein binding assay mix containing 12 mM HEPES (pH 7.9), 12% glycerol, 60 mM KCl, 5 mM MgCl₂, 0.12 mM EDTA, 0.3 mM DTT, 0.1 μg poly (dI/dC), and 0.05 μg of denatured salmon sperm DNA as carrier was used for EMSA. For detecting the super shift, K-RTA or K8 nuclear extracts were incubated with anti K-RTA or anti-K8 antibody along with the labeled probe. The reaction mix was incubated on ice for 30 mins and the bound complexes were resolved on 5% non-denaturing polyacrylamide gel in 0.5X TBE buffer (0.045 M Tris Borate, pH 8.2, and 1.0 mM EDTA). The gel was run at 4°C at 100V for 10–12 h and then exposed overnight on a phosphoimager screen. The signals were detected by autoradiography (GE Healthcare, Pittsburgh, PA).