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. 2005 May 26;102(23):8156–8161. doi: 10.1073/pnas.0503402102

Fig. 5.

Fig. 5.

Gel-filtration chromatography of TrwBΔN70 and TrwBΔN70–DNA complexes. Protein samples (60 μM) were preincubated with oligonucleotides of different lengths (10 μM) and chromatographed at 20°C at a flow rate of 30 μl/min on a Superdex-200 PC 3.2/30 column on a Amersham Pharmacia SMART system. The column was equilibrated in buffer containing 50 mM Pipes·NaOH (pH 6.0), 100 mM NaCl, 2 mM MgCl2, 0.1 mM EDTA, and 10% glycerol. Absorbance was monitored at 280 nm (black) and 260 nm (red). Ve represents the elution volume. Elution profiles are as follows: TrwBΔN70 (A); TrwBΔN70 preincubated with oligonucleotides 29-, 36-, or 40-mer (B); and TrwBΔN70 preincubated with oligonucleotides 45- or 66-mer (C). Peaks were interpreted as follows: (1) free protein (relative lack of absorbance at 260; apparent molecular mass = 50 kDa), (2) slow complex (contains protein and DNA; apparent molecular mass = 100 kDa), and (3) fast complex (contains protein and DNA; apparent molecular mass = 440 kDa).