Figure 5.

Trypsin digestion of Cbp2_Hb and the Cbp2_Hb–CRISPR-2r_Hb complex. (A) Six microgram Cpb2_Hb were incubated with trypsin (specific activity: 806 USP U/mg) in 10 mM phosphate (Na₂HPO₄/KH₂PO₄), 137 mM NaCl, 2.7 mM KCl, pH.7.4 for 15 min at 37°C and electrophoresed and stained as in Figure 2A. Lane 1—protein size ladder, lane 2—Cbp2 without trypsin and lanes 3 and 4—treated with 12 and 60 ng of trypsin, respectively. Arrows indicate undegraded and degraded Cbp2_Hb. The weak upper bands in lane 2 probably represent Cbp2 oligomers, as in Figure 2A. (B) The Cpb2_Hb–CRISPR-2r_Hb complex (8 nm [³²P] 5′-end labelled DNA per sample) was formed at a 4:1 protein:DNA molar ratio and treated with increasing concentrations of trypsin. Lane 1—CRISPR-2r_Hb, lane 2—Crp2_Hb DNA complex, lanes 3 and 4—complex was incubated with 45 and 90 ng trypsin, respectively. Trypsin digestions were performed in 15 mM (NH₄)₂CO₃, 10 mM Mg acetate, pH 7.9 for 15 min at 37°C. Samples were analysed by electrophoresing in 12.5% polyacrylamide gels and autoradiographed (see Materials and Methods).