Proteins were eluted with a constantly increasing gradient between the lysis buffer and 0.75 M imidazole, 20 mM NaPO4, 0.5 M NaCl,

pH = 7.4. Proteins were then dialyzed against 1 × e0 buffer (50 mM Tris [pH = 7.5], 1 mM dithiothreitol, 1 mM phenylmethanesulfonyl fluoride, and 100 μl/l Tween-20). Glycerol was added to a final concentration of 10% (vol/vol), and aliquots were snap frozen in liquid nitrogen and Selleck Birinapant stored at -80°C. Purity of protein preparations was assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by staining with Coomassie brilliant blue. BCA (bicinchoninic acid) protein assays (Pierce, Rockford, IL), calibrated with bovine serum albumin (Pierce), were used to determine protein concentrations. Electrophoretic mobility shift assays (EMSA) All EMSAs were performed at least three times. Biotin-labeled DNA probes were produced based upon the sequence of the B. burgdorferi strain B31 erpAB 5′-noncoding DNA, to which the orthologous EbfC protein is known to bind [7, 8, 10]. Probe b-WT corresponds with bp -160 through -36 (relative to the start

of TPX-0005 cell line translation) of the erpAB operon, and contains two consensus EbfC-binding sites [8, 10] (Fig. 2). Probe b-WT selleck products was produced by PCR using oligonucleotide primers bio-A14A (5′-biotin-TTGTAATGAGTAGTGCATTTG-3′) and R8 (5′-GCAATATTTCAAAGATTTAAA-3′) from DNA template pBLS591 [7]. That same oligonucleotide primer pair was used to produce probe b-C2 from mutant template pSRJ-2, a derivative of pBLS591 in which EbfC-binding site II was changed to CACAACA (Fig. 2) [10]. Probes b-C20, b-C30, b-C40 and b-C50 were also produced using primers bio-A14A and R8, from mutant templates pSRJ-20, pSRJ30, pSRJ40 and pSRJ50, respectively, selleck chemicals derivatives of pSRJ-2 in which single bp mutations were introduced to site I (Fig. 2) [10]. Each PCR reaction product was separated by agarose

gel electrophoresis and DNA visualized by ethidium bromide staining. Amplicons were extracted from gels into nuclease-free water using Wizard SV (Promega, Madison, WI), and quantified by spectrophotometric determination of absorbance at 260 nm. EMSAs were performed using 100 pM biotin-labeled DNA fragment and varying concentrations of purified recombinant YbaBEc or YbaBHi. Binding conditions consisted of 50 mM Tris-HCl (pH = 7.5), 1 mM dithiothreitol, 8 μl/ml protease inhibitor (Sigma-Aldrich, St. Louis, MO), 2 μl/ml phosphatase inhibitor cocktail II (Sigma-Aldrich), and 10% glycerol. Protein and DNA were mixed together, in final volumes of 10 ml, and allowed to proceed toward equilibrium for 20 minutes at room temperature, then subjected to electrophoresis through 6% DNA retardation gels (Invitrogen) for 9000 V-min. DNA was electrotransferred to Biodyne B nylon membranes (Pierce), cross-linked by ultraviolet light, and biotinylated DNA detected using Chemiluminescent Nucleic Acid Detection Modules (Pierce).