Susceptibility testing Oxacillin resistance levels were compared by swabbing 0.5 McFarland cell Cyclopamine molecular weight suspensions across agar plates containing appropriate concentration gradients of oxacillin. For population analysis profiles, appropriate dilutions of an overnight culture, ranging from 100 to 108, were plated on increasing concentrations of oxacillin. Plates were incubated at 35°C and colony forming units per ml (cfu/ml) were determined after
48 h. Binding-protein purification Crude protein extracts were isolated from CHE482, grown under normal culture conditions until OD600 DAPT cost nm 1.5. Cells were harvested, resuspended
in PBS (pH 7.4) and mechanically lysed using Lysing Matrix B (BIO 101 Systems) tubes and a FastPrep FP120 (BIO 101 Systems). Suspensions were clarified by centrifugation and supernatants, containing soluble cytoplasmic proteins, were transferred to Amicon Ultra centrifugal filter devices (Millipore) with a pore cut-off size of 10 kDa. Proteins were then washed and concentrated in 1× binding buffer (10 mM Tris-HCl, pH 7.5, 1 this website mM EDTA, and 1 mM DTT, 0.5 M NaCl). Protein concentrations were measured by Bradford assay (BioRad Laboratories GmbH) . Primer pair me36F/me36Rbiot (Table 2) were used to amplify a biotinylated mecA
promoter/operator fragment, which was bound to streptavidin coated magnetic beads (Dynabeads M-280 Streptavidin, DYNAL BIOTECH) according to the manufacturer’s instructions. Binding reactions, containing DNA-coated beads mixed with 100 μg of crude protein extract in 1× protein binding buffer (20 mM Hepes, pH 7.6, 1 mM EDTA, 10 mM (NH4)2SO4, 1 mM DTT, 0.2% Tween 20 (w/v), 30 mM KCl), 0.02 μg/μl poly d(I-C) and 2 ng/μl poly L-lysine, were incubated at room temperature for 30 min with constant rotation. Beads were then washed and binding-proteins eluted in elution buffer (1× protein binding buffer containing 2 mafosfamide M KCl). Eluted proteins were dialysed against water, concentrated by evaporation, and run on 15% SDS polyacrylamide gels. Gels were silver stained using the Protein Silver Staining kit (Amersham Biosciences AB) without the addition of glutaraldehyde. Protein bands were excised from gels and analysed by mass spectrometry (LC/ESI/MS/MS) at the Functional Genomics Centre, Zurich. The SA1665 protein sequence [BAB42933] was analysed by Blast search http://www.ncbi.nlm.nih.gov/BLAST and motif scan http://myhits.isb-sib.ch/cgi-bin/motif_scan. Table 2 Oligonucleotide primers used in this study.