polymorphic DNA experiments were replicated three times to ensure reproducibility of the assay. The PCR mixture contained 60 mM Tris–HCl, pH 8.5, 15 mM (NH4)2SO4, 2 mM MgCl2, 0.125 mM each of dATP, dCTP, dGTP, and dTTP, 7.5 picomoles of a single 10mer, 4 μl of cell suspension, and 0.625 units of Taq polymerase (Applied Biosystems, Foster City, CA). Controls containing no H. parasuis cells were also included. Amplification of DNA was performed on a GeneAmp PCR System 9600 (Perkin Elmer, Boston, MA). Cells were lysed in a “hot start” step  at 94°C for 10 min, and then amplified for 45 cycles of 1 min at 94°C, 1.5 min at 36°C, and 2 min at 72°C, followed by an extension step for 10 min at 72°C, then a hold step at 4°C. PCR products were stored at −20°C, until they were analyzed on 1% agarose horizontal gels in Tris-Borate-EDTA (TBE), pH 8.3 buffer AZD9291 in vitro  and detected by ultraviolet light illumination after staining with ethidium bromide. The DNA standard was a 1 kb ladder (Invitrogen, Carlsbad, CA). SDS-PAGE analysis For WCP lysates, bacterial cells grown in Frey’s broth for 22 h were pelleted by centrifugation at 675 × g for 10 min. Cells were washed in 0.1
M phosphate buffered saline (PBS), pH 7.2, containing 1 mM Pefabloc (Roche Diagnostics, Indianapolis, IN), then resuspended at a ratio of 32 mg cells per 100 μl PBS/Pefabloc. selleck kinase inhibitor Cells were sonicated with a GANT61 cell line microprobe (Heat Systems-Ultrasonics, Farmingdale, NY) at 50% power for 60 1-second bursts to lyse them and centrifuged at 16,000 × g for 20 min to remove cell debris. Protein concentrations were determined by the Folin-Lowry method  with bovine serum albumin as a standard. Protein (10 μg/well) was applied to 10-well Ureohydrolase NuPAGE precast
4-12% gradient Bis-Tris gels (Invitrogen). NuPAGE antioxidant (Invitrogen) was used in 3-(N-morpholino)-propane sulfonic acid (MOPS) running buffer (Invitrogen). The protein prestained standard was BenchMark, 10–200 kDa (Invitrogen). Running conditions were 10 mA/gel for 15 min, then 200 V for 40 min. Gels were stained in 0.1% Coomassie Brilliant Blue R250 in 50% methanol/10% acetic acid and destained in 50% methanol/10% acetic acid. Electrophoresis pattern analysis Gels were photographed, scanned (Kodak Image Station, Rochester, NY) and the image was digitized (Kodak Molecular Imaging Software, New Haven, CT). RAPD and protein profiles were analyzed using Gel Compar II software (Applied Maths, Austin, TX). Bands were coded as binary data (absent = 0 or present =1), regardless of band intensity. Optimal settings for band optimization and band position tolerance levels were calculated for each primer. Primer 2 values were 2.16% for band optimization and 4.72% for band position tolerance. Similarly, primer 7 values were 1.23% and 1.06%, while primer 12 values were 0.34% and 0.72%, respectively.