= three separate experiments and for each three different fields were measured). from this venom and demonstrate this to be a selective inhibitor of collagen-induced functions of platelets and endothelial cells. EXPERIMENTAL Methods Protein Purification Venom of was extracted from several specimens managed in the herpatarium of the Alistair Reid Venom Study Unit (Liverpool School of Tropical Medicine) and freezing immediately and lyophilized. Ten milligrams of the pooled, lyophilized venom was dissolved in 2 ml of 20 mm TrisCl, pH 7.4, and centrifuged at 13,225 for 3 min to remove the insoluble parts. The obvious venom sample was loaded on to a 1-ml prepacked Q-Sepharose anion-exchange column (GE Healthcare). The column was washed with 10 column quantities of 20 mm TrisCl, pH 7.4, to remove the unbound proteins before 500-l fractions were collected having a 0C50% of 1 1 m NaCl linear gradient over 20 column quantities at a rate of 0.5 ml/min. Fifty microliters of selected fractions were analyzed by 10% non-reducing SDS-PAGE. The fractions with target protein were diluted to reduce the salt concentration and rerun on Q-Sepharose to increase purity. Selected fractions were further purified by gel filtration chromatography (Superdex 75) and were analyzed using 10% non-reducing SDS-PAGE. The fractions with target protein in pure form were concentrated by ultra-filtration (5-kDa cut-off). The amount of protein purified was estimated using RC DC protein assay kit (Bio-Rad). Ten micrograms of genuine protein was analyzed using 4C20% reducing and non-reducing SDS-PAGE gradient gel (Bio-Rad) and 10C20% Tris-Tricine gel (Bio-Rad) to analyze the dimeric nature of the protein. Sequencing and Mass Spectrometry Analysis of Purified Protein Ten micrograms of purified protein was separated using 10C20% Tris-Tricine gel and immobilized on a PVDF membrane and subjected to Edman degradation method for sequencing. The trypsinized gel slices comprising purified venom protein were subjected to LC-MS/MS mass spectrometry analysis as explained previously (24), and the peptide sequences were analyzed by EMBOSS pairwise sequence alignment (26) and ClustalW2 multiple sequence alignment (27). PCR Amplification, Cloning, and Sequencing of Amplified DNA A primer (5-ATGGGGCGATTCATCTTCC-3) complimentary to the 5 transmission peptide coding sequence of the known C-type lectin-2 sequence (NCBI accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AY429478″,”term_id”:”37993392″AY429478) (28) was synthesized by Sigma Aldrich. This VCP-Eribulin usband the M13 ahead (5-GTAAAACGACGGCCAGTC-3) primer were used to amplify snaclec genes in the previously constructed (24) venom gland cDNA library of by PCR (30 cycles) using denaturation at 94 C for 30 s, annealing at 55 C for 30 s, extension at 72 C for 1 min and a final extension at 72 C for 10 min. Amplicons were analyzed by 1% (w/v) agarose gel electrophoresis and purified from your gel using the Wizard? SV Gel and PCR Clean-up System (Promega). Eluted DNA was cloned into a pGEM T-Easy Cloning system (Promega) according to the manufacturer’s protocols and used to transform was confirmed to also bind rhinocetin from venom and therefore was used to detect rhinocetin in subsequent circulation cytometry and immunoblotting experiments. Mouse monoclonal antibodies raised against the 2 2 Rabbit Polyclonal to T3JAM subunit of integrin 21 (32) (clone 6F1, from professor B. S. Coller, Rockefeller University or college) were used in circulation cytometry. The secondary antibody for immunoblotting (Cy3? goat anti-mouse IgG) was from Invitrogen. Circulation Cytometry Analysis Platelet-rich plasma (PRP) diluted 10-collapse in HEPES-buffered saline VCP-Eribulin was incubated with numerous dilutions of the mouse anti-snaclec serum and Cy3?-labeled goat anti-mouse IgG in a total of 50 l of volume for 20 min at room temperature. Cells were then fixed with VCP-Eribulin 450 l of 0.2%.