Figure 5

Electrophoretic mobility shift assays using purified and partially purified preparations of the IS 2 OrfAB-GFP fusion protein. (A) Purified OrfAB-GFP fusion protein preparations shown in Figure 4c and the purified native protein from refolding experiments were used in gel retardation reactions. 0.46 μM of the fusion protein and 6.02 μg of the refolded protein were reacted for 30 minutes at room temperature (20°C) with 2 nM of ³²P-labeled annealed 87-mer oligonucleotides containing the 41 bp inverted right repeat sequence. The reactions were run at 4°C at 120 mA for 2400 Vhr in a 5% native polyacrylamide gel. The arrow shows complexes formed with low efficiency. Lanes: 1. Protein-free control. 2. Refolded native OrfAB. 3. OrfAB-GFP. (B) Partially purified preparations of the OrfAB-GFP fusion protein shown in Figure 4a and crude extracts from overexpression of the pTW2 OrfAB-GFP construct used in binding reactions. Approximately 80 nM of the protein from the partially purified preparations shown in Figure 4a and from the crude extracts were reacted with 2 nM of the ³²P-labeled annealed 87-mer oligonucleotides as described in part A. The reactions were run for 1400 Vhrs at 4°C. Lanes: 1. Protein-free control. 2. Partially purified preparation of OrfAB-GFP. 3. Crude extract from the overexpressed pTW2 OrfAB-GFP plasmid. Bp: base pairs; GFP: green fluorescent protein; orf: open reading frame; Vhr: volt hour.