P. Tucker, Nicholas G. Hicks, Matthew A. Clarke, Thomas Crack, Jason C. Chandra, Govind E. Le Brun, Nick Dixon, Ray I. Hutchings, Matthew DNA binding assays with the <i>hmpA1</i> and <i>hmpA2</i> promoters and purified NsrR protein. <p>(A). Bandshift assay using 200 base pair restriction fragments (20 ng per reaction) carrying the <i>hmpA1</i> and <i>hmpA2</i> promoters, as indicated, and purified [2Fe-2S] NsrR in 50 mM Tris pH 7.0, 100 mM NaCl buffer or 50 mM Tris pH 7.0, 100 mM NaCl buffer saturated with NO (2 mM), as indicated. Binding is abolished by addition of NO saturated buffer to purified NsrR. (B). Bandshift assay using a 200 base pair restriction fragment (20 ng per reaction) carrying the <i>hmpA1</i> promoter with either EDTA∶ferricyanide treated apo-NsrR or holo [2Fe-2S] NsrR as indicated in 50 mM Tris pH 7.0, 100 mM NaCl buffer. The apo-form of the protein is unable to bind to the <i>hmpA1</i> promoter indicating that the cluster is required for DNA binding activity. (C). Sedimentation equilibrium of oligonucleotides in the presence and absence of purified NsrR. The sedimentation of each sample was monitored at 260 nm and fitted to a single component model as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003623#s3" target="_blank">Materials and Methods</a>. (Left) Lower panel: absorbance profile of 2 µM <i>hmpA2</i> and 10 µM NsrR in 50 mM Tris-HCl pH 7.0, 100 mM NaCl after centrifugation at 16,000 (□), 18,000 (O) and 20,000 (Δ) rpm. Upper panel: Residual profile of the difference between the data and fitted curves. (Right) Lower panel: absorbance profile of 2 µM <i>hmpA2</i> in 50 mM Tris-HCl pH 7.0, 100 mM NaCl after centrifugation at 16,000 (□), 18,000 (O) and 20,000 (Δ) rpm. Upper panel: Residual profile of the difference between the data and fitted curves.</p> binding;assays;promoters;purified;nsrr 2013-02-21
    https://plos.figshare.com/articles/figure/_DNA_binding_assays_with_the_hmpA1_and_hmpA2_promoters_and_purified_NsrR_protein_/586968
10.1371/journal.pone.0003623.g003