0, 300 mM NaCl, 25 mM imidazole, and 5 mg/ml lysozyme and incubated on ice for 30 min. Subsequently, the cells were further RepSox molecular weight lysed by sonification (4 × 1 min pulse, 1 min break, MS72 probe with 25% power; Bandelin Sonoplus HD2200, Berlin, Germany) and the soluble 6His-MleR extract was separated from insoluble cell material by centrifugation (25,000 × g, 30 min, 4°C). The 6His-MleR protein was then purified by IMAC affinity chromatography using Talon resin (Clontech, Saint-Germain-en-Laye, France). Bound protein
was washed with 8 bed volumes 50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 25 mM imidazole and eluted with 50 mM NaH2PO4, pH 7.0, 300 mM NaCl, 300 mM imidazole. The eluted 6His-MleR protein (purity >90% on an SDS PAGE) was always stored on ice and was verified by western blot (Anti His-tag antibody, Novagen) and N-terminal sequencing. Electrophoretic mobility shift assay (EMSA) For binding studies, the purified MleR protein was dialysed four times against 1 liter 1× binding buffer (20 mM Tris, pH 7.5, 100 mM KCl, 2 mM EDTA, 10% glycerol) at 4°C for 12 hours using a
12-14 kDa cut-off dialysis bag (Medicell International Ltd., London, UK). Several fragments of the region between mleR and mleS were PCR amplified and directly used for gel retardation experiments (see Table 3 for primers). To verify the specificity of the DNA-MleR interaction each KU-57788 cell line reaction mixture contained an equal amount of competitor DNA. Competitor DNA consisted either of an internal fragment of mleS, amplified by PCR (primers 137qF/R), or a DNA fragment within the upstream region of mleR, generated by hybridising complementary primers (EP10/11, Gemcitabine cell line Table 3). Nepicastat solubility dmso For this purpose, primers EP10/11 were mixed in equal molar ratios, denaturated by heating to 100°C and annealed by slowly cooling down to room temperature. DNA fragments, MleR protein (appr. 100 ng) and competitor DNA (in case of the complementary primers 75 ng/μl, final concentration) were mixed and incubated for 20 min at ambient temperature. To further exclude unspecific interactions, MleR was substituted with 100 ng BSA (Carl-Roth) and tested for each fragment. The reaction mixtures
were subsequently loaded onto a 0.5× TBE, 4.5% polyacrylamide (37.5:1, acrylamide/bisacrylamide) gel. Since the MleR protein has a calculated pI of ~9, DNA in complex with MleR was hardly entering the gel using pH values below 9.2. Therefore the pH of the gel cast solution and electrophoresis buffer were adjusted to pH 9.45. L-malate was added to the binding reaction, the gel and the electrophoresis buffer (0.5× TBE) at 5 mM final concentration when needed. Electrophoresis was carried out at 10 V/cm at ambient temperature and the gel was stained using SYBR Gold (Invitrogen). Acknowledgements We would like to thank Andreas Podbielski for providing the pFW5 plasmid and Holger Lössner for providing the pHL222 plasmid. References 1.