Trans blot turbo transfer semi dry transfer system

Band-shift experiments were performed by incubating 20 fmol of biotin labeled double stranded oligos with recombinant human ELF1 protein (Origene, TP760629) or recombinant GABPA/B (Abnova, GABPA: H00002551-P01, GABPB: H00002553-P01) in a total volume of 20 L of protein binding buffer (PBB: 150 mM NaCl, 0.25% NP40, 50 mM TRIS, pH 8.0, and 1 mM DTT) for 30 min. For GABP experiments, GABPA and GABPB were mixed at equimolar concentrations for 20 min at room temperature prior to addition of the oligo, in GABP experiments, the molecular weights listed in the figures refers to the total molecular weight of protein used (GABPA/B combined). The resulting protein complexes were resolved on 4-20% TBE gels (Biorad) in a Mini-PROTEAN tetra cell (Biorad) at 100 V for approximately 3 h in 1X TBE. Samples were transferred onto a nylon membrane (Biodyne) in a Trans-Blot Turbo Transfer semi-dry transfer system (Biorad) at 400 mA for 10 min. Membranes were UV cross-linked and oligos were detected using streptavidin-HRP conjugate and a chemiluminescent substrate (Chemiluminescent Nucleic Acid Detection Module, Pierce). Raw data from all bandshift experiments reported in this study is presented in Supporting Information Fig. 1.