015 M Tris–HCl, pH 7 95, until bands of activity become clear Th

015 M Tris–HCl, pH 7.95, until bands of activity become clear. The protein molar mass standards were always separated at the extreme end of the gel plate and following electrophoresis, the line was carefully sectioned and stained with Coomassie brilliant blue R-250. CK and CK–MB levels in the serum of envenomed rats were determined as a measured of the cardiotoxicity of H. lunatus venom. Groups of six Wistar rats were injected intraperitoneally (i.p.) with 750 μg of

soluble this website venom or ultra-pure water (control). The animals were kept under inhalation anesthesia with morphine (2.5 mg/kg) and diazepam (2.5 mg/kg), injected via the intramuscular route ( Flecknell et al., 1996). After 30 min of envenoming, blood was collected by cardiac puncture. Blood was then centrifuged (3000 rpm for 5 min) and serum used for biochemical analysis. The levels of

creatine kinase isoenzyme MB (CK–MB) and total creatine kinase (CK) were measured using commercial kits from Bioclin (Quibasa, Brazil) and a Thermo Plate Analyzer Basic instrument. Chromatographic fractionation of H. lunatus venom was performed using high performance liquid chromatography (HPLC). Briefly, 1 mg of crude venom was applied to a reverse phase column. The column used in this assay was a Shimadzu-Pack CLC-ODS C18 (6 × 150 mm) eluted at 1 mL/min with a linear gradient of 0.1% TFA in water and acetonitrile, solutions A and B, respectively. After column equilibration the venom fractions NVP-BKM120 were Buspirone HCl separated with a linear gradient from

solution A to 60% solution B, running for 60 min. Fractions were then subjected to MALDI-TOF-TOF analyses. MS analysis was performed using a MALDI-TOF-TOF AutoFlex III™ (Bruker Daltonics) instrument in positive/reflector mode controlled by the FlexControl™ software. Instrument calibration was achieved by using Peptide Calibration Standard IV (Bruker Daltonics) as a reference and using sinapinic acid as a matrix. The peak was spotted to MTP AnchorChip™ 400/384 (Bruker Daltonics) targets using standard protocols for the dried droplet method. Adult New Zealand female rabbits were used for the production of anti-H. lunatus and anti-T. serrulatus venom antibodies. After collection of pre-immune sera, the animals received an initial subcutaneous injection of 100 μg of whole venom in complete Freund’s adjuvant (day 1). Three booster injections were made subcutaneously 14, 21 and 28 days later with a lower dose (50 μg) in incomplete Freund’s adjuvant. The animals were bled one week after the last injection. Maxisorp microtitration plates (Nalge Nunc, USA) were coated overnight at 5 °C with 100 μL of a 10 μg/mL solution of H. lunatus, T. serrulatus, A. australis or C. sculpturatus whole venom in carbonate buffer pH 9.6. After blocking (3% powdered milk in PBS) and washing (0.05% Tween-saline), sera from pre-immune and immune rabbit were added in different dilutions and incubated for 1 h at 37 °C.

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