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Protein-inhibitor complex was prepared by incubating 50 M of the 3CL-GP-6xHis with 500 M of inhibitor in a buffer comprised of 50 mM Tris-HCl (pH 7.5), 1 mM EDTA for 1 h at 4 C before immobilization onto beads

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After the rst chlorine reacts, the second requires a slightly higher temperature for its reaction (20C), and the third chlorine needs an even higher temperature (80C)

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