Sequence reagents and all other reagents and chemicals were from

Sequence reagents and all other reagents and chemicals were from Calbiochem-Merck (Darmstadt, Germany). Tetravalent anti-bothropic (B. jararacussu, Bothrops jararaca, find more Bothrops neuwiedi and Bothrops alternatus) and monovalent anti-crotalic (C. d. terrificus) horse antivenom were produced and kindly provided by the Vital Brazil Institute, Niteroi, RJ, Brazil. Two libraries of sixty-nine, 14-mer peptides were designed to represent

a consecutive overlapping coverage that was offset by nine amino acids across the entire coding region (121–122 amino acids) of the three PLA2s present in the venom of B. jararacussu. Sequences were obtained from the UniProtKB – Protein knowledgebase (http://www.uniprot.org/): BthTX-I (Swiss-Prot ID.: Q90249), BthTX-II (Swiss-Prot ID.: P45881) and BthA-I (Swiss-Prot ID.: Q8AXY1). The peptides were automatically prepared onto Amino-PEG500-UC540 cellulose membranes according to standard SPOT synthesis protocols ( Frank, 2002) using an Auto-Spot Robot ASP-222 (Intavis Bioanalytical Instruments AG, Köln, Germany). In brief, coupling reactions were followed by acetylation

with acetic anhydride (4%, v/v) in N, N-dimethylformamide to render peptides unreactive during the subsequent steps. Enzalutamide After acetylation, Fmoc protective groups were removed by the addition of piperidine to render nascent peptides reactive. The remaining amino acids were added by this same process of coupling, blocking and deprotection until the expected desired peptide was generated. After the addition of the last amino acid in the peptide, the amino acid side chains were deprotected

using a solution of dichloromethane–trifluoracetic acid–triisobutylsilane (1:1:0.05, v/v/v) and washed with methanol. Membranes containing the synthetic peptides were either probed immediately or stored at −20 °C until needed. Negative controls [without peptide; IHLVNNESSEVIVHK (Clostridium tetani) precursor peptide] and positive controls were included in each assay. SPOT membranes were washed with Cyclin-dependent kinase 3 TBS (50 mM Tris-buffer saline, pH 7.0) and blocked with TBS-CT (50 mM Tris-buffer saline, 3% casein, 0.1% Tween 20, pH 7.0) at room temperature under agitation or overnight at 4 °C. After extensive washing with TBS-T (50 mM Tris-buffer saline, 0.1% Tween 20, pH 7.0), two membranes presenting the same peptide library were incubated separately for two hours with either horse anti-crotalic or anti-bothropic antivenom (1:250) in TBS-CT and them washed again with TBS-T. Afterward, the membranes were incubated with alkaline phosphatase-labeled sheep anti-horse IgG (1:5000 in TBS-CT) for one hour, and then washed with TBS-T and CBS (50 mM citrate-buffer saline, pH 7.0). Chemiluminenscente CDP-Star® Substrate (0.25 mM) with Nitro-Block-II™ Enhancer (Applied Biosystems, USA) was added to complete the reaction. Chemiluminescent signals were detected on MF-ChemiBis 3.2 (DNR Bio-Imaging Systems, Israel) at a resolution of 5 MP.

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