, 2011). Bacteria were grown in CSE minimal medium supplemented with 0.5% of the indicated carbon source. At the time of harvest, the pH of the culture www.selleckchem.com/products/Erlotinib-Hydrochloride.html medium was lowered to pH 4.5 using 12 M HCl to stop HPrK/P activity, as described previously (Singh et al., 2008). Aliquots of 45 mL culture were pelleted by centrifugation, washed with 500 mM NaCl and 20 mM sodium acetate pH 4.5, and finally re-suspended in 1 mL of the same buffer. Cells were disrupted by three passages through a French pressure cell. All protein
extracts were separated by non-denaturing gel electrophoresis (100 V, 75 min) using gels prepared from 12% acrylamide in 375 mM Tris/HCl pH 8.8. Crh and HPr were subsequently detected by Western blotting. For the determination of total Crh and HPr amounts, SDS sample buffer [62.5 mM Tris/HCl pH 6.8, 5% (v/v) β-mercaptoethanol, 2% (w/v) SDS, 10% (v/v) glycerol, 0.05% (w/v) bromophenol-blue] was added to protein extracts. Ixazomib concentration The samples were heated for 10 min at 70 °C and subsequently separated by SDS-PAGE using 15% (Fig. 1) or 12% polyacrylamide gels (Figs 2 and 3) prepared in 375 mM Tris/HCl pH 8.7 and 0.1% SDS. Gels were analyzed by Western blotting. To identify an epitope in Crh
hat was suitable for its specific detection, a sequence alignment of various Crh proteins and their cognate homologs was performed using clustalw software. This analysis showed that the primary sequences of the Crh proteins deviate considerably from the HPr sequences in three regions. Subsequent analysis of the secondary structures of these regions (Janin, 1979; Parker et al., 1986) suggested the 23 C-terminal amino acids of Crh as an epitope suitable for the generation of an antiserum
that allows specific detection of Crh and does not cross-react with HPr. Consequently, this peptide, which additionally carried a cysteine at the N-terminus for its coupling to a protein carrier, was synthesized and used for the immunization of rabbits (performed by Pineda, Antikörper Services, Berlin, Germany). After gel electrophoresis, proteins were transferred to a polyvinylidene difluoride membrane (Bio-Rad) by electro-blotting (60 min at 0.8 mA cm−2). Polyclonal rabbit antisera directed against Arachidonate 15-lipoxygenase HPr (Monedero et al., 2001) or the C-terminus of Crh (this work) were used at dilutions of 1 : 10 000 to detect these proteins. The primary antibodies were visualized with goat anti-rabbit IgG secondary antibodies conjugated to alkaline phosphatase diluted 1 : 100 000 (Promega) and the CDP* detection system (Roche Diagnostics). Quantification of signal intensities was achieved using software imagej version 1.42 (Abramoff et al., 2004). A useful technique that allows the investigation of the phosphorylation state of a protein in vivo involves the separation of the differently modified protein species by non-denaturing polyacrylamide gel electrophoresis (PAGE) followed by their sensitive detection by Western analysis.