Analysis of OPBTc under SDS–PAGE and size exclusion chromatography.

<p>(A) 2 µg of OPBTc was subjected to 10% SDS–PAGE with buffer loader containing 4% (1), 1% (2), 0.5% (3), 0.1% (4) or 0.05% (5) final SDS concentration without DTT or (B) with 10 mM DTT. (C) OPBTc (500 ng) <i>in gel</i> activity was detected by incubating a replica of the gel in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030431#pone-0030431-g001" target="_blank">Fig. 1B</a> with the fluorogenic substrate <i>N</i>-CBZ-Gly-Gly-Arg-AMC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030431#s2" target="_blank">Material and Methods</a>. (D) Size Exclusion Chromatography of OPBTc was followed by activity assay at pH 8.0. The dotted black line with circles indicates the elution volumes of reference proteins. A.U. means arbitrary fluorescence units.