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. 2011 Jul 9;39(19):8488–8502. doi: 10.1093/nar/gkr553

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — (A) SDS–PAGE analysis of cross-linked GyrB. W661C–GyrB^nc, T546C–GyrB^nc and A543C–GyrB^nc (4 µM) were incubated under non-reducing conditions alone or in the presence of BMH (25 µM) or (1,10-phenanthroline)copper(II) (CuP, 125 µM). Reactions were incubated for 1 h at 37°C for BMH cross-linking and quenched with DTT (4 mM). Reactions were incubated for 0.5 h at 37°C for (1,10-phenanthroline)copper(II) cross-linking and quenched with EDTA (1 mM). The above quenched mixtures were analyzed by SDS–PAGE under non-reducing conditions. T546C–GyrB^nc protein was purified with gel filtration (B) and its peak II was cross-linked by (1,10-phenanthroline)copper(II) (C). Lane 1, protein marker; lane 2, the prepared cross-linked T546C–GyrB^nc mutant protein by (1,10-phenanthroline)copper(II); lanes 3 and 4 were the cross-linked protein incubated with 5 and 10 mM DTT, respectively. Samples were analyzed by SDS–PAGE under nonreducing conditions.