Simplybluetm safestain

For the proteome analysis (Figure 1), 10 μg of protein extract were used for a short migration 1D gel electrophoresis (NuPAGE^® 4-12% Bis-Tris Gel, novex). Proteins were visualized using Coomassie G-250 (SimplyBlue^TM SafeStain, Invitrogen) and the whole colored part of each lane was cut into small pieces. The gel pieces were destained using Solvent A (10% v/v acetic acid, 40% v/v ethanol) and Solvent B (50% v/v 50 mM ammonium bicarbonate, 50% v/v acetonitrile). The proteins contained in the gel were reduced by 10 mM dithiothreitol (Sigma) and alkylated by 55 mM iodoacetamide (Sigma). The proteins were digested with 200 ng trypsin (Promega) and afterwards extracted using a solution of 0.5% v/v trifluoroacetic acid and 50% v/v acetonitrile. The peptides were dried completely using a concentrator (Savant^TM SPD121D, Thermo Fisher Scientific) and taken up in 50 μl loading buffer (0.08% v/v trifluoroacetic acid, 2% v/v acetonitrile) for LC-MS/MS proteome analysis (4 μl = 800 ng peptide per injection).

Purity of scFvs was determined by SDS-PAGE with 2.5–5 µg protein and subsequent staining of proteins with SimplyBlue^TM SafeStain (Invitrogen). For immunoblot analysis 1.5 µg of scFv were separated by SDS-PAGE electrophoresis under reducing conditions. After protein transfer to a PVDF membrane (Whatman), scFvs were detected using a primary mouse anti-c-myc antibody (Invitrogen) and a secondary polyclonal rabbit anti-mouse IgG HRP conjugate (Dako). Biotinylated scFv-BAPs were detected with a HRP-conjugated anti-biotin antibody (Sigma-Aldrich). Visualization of scFvs was performed by Luminata Forte Western HRP substrate (Merck Millipore) and LAS 3000 chemoluminescence imager (FujiFilm). For analysis of scFv-BAP complexation with avidin, the two components were mixed at various molar ratios ranging from 1:2 to 8:1 in PBS. After 30 min incubation at room temperature scFv-BAP-avidin conjugates as well as non-conjugated biotinylated scFv-BAPs were analyzed by non-reducing SDS-PAGE and detected by Western blot analysis as described above.

Fibrinogen (200 ng, Enzymes Research, UK) was separated on 10 % Bis-Tris NuPAGE Gels at 180V for 75 min (ThermoFisher Scientific) with colourmetric protein markers (BioRad Kaleidoscope). The gel was split and a portion was stained with SimplyBlueTM SafeStain (Invitrogen) to determine the positions of the α, β and γ chains. The other portion was transferred in 20 % methanol to PVDF (1 h, 100 V). The membrane was subsequently blocked [5 % (w/v) BSA in PBS-T] overnight at 4 °C, probed with 500 nM rFnBPB or rFnBPB_N277A/F279A in 5 % (w/v) BSA PBS-T for 4 h 20 °C. The membrane was washed three times in PBS, before application of Anti-His₆-Peroxidase (Roche, 1 : 1000) in 5 % (v/w) BSA PBS-T. The membrane was washed three times in PBS, before 1 min incubation in 1×LumiGLO/1×Peroxidase (CellSignal). Blots were visualised in an Amersham A680 imager.

IgG was isolated with the Melon^TM Gel IgG Spin Purification Kit (Thermo Scientific^TM 45,206) from human and murine plasma by diluting 15 µL plasma with 95 µL of purification buffer and isolation according manufacturer’s instructions. Antibody concentration was determined as the means of A280 duplicate measurements (Epoch Take3 system) and the integrity of antibodies was determined via gradient sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (NuPAGE^TM 4–12% Bis-Tris (Invitrogen^TM NO0336) in 1X MOPS (Invitrogen^TM NP0050)) and subsequent Coomassie staining (Invitrogen^TM SimplyBlue^TM SafeStain). Two µg of eluate was mixed with 2.5 µL 4X LDS buffer (Pierce^TM 84788) and filled with buffer to 10 µL, denatured at 70 °C for 10 min and loaded to each lane and gel run at 180 V for 60 min. IgG was concentration adjusted to 0.3 mg/mL (human) and 0.2 mg/mL (murine) with the kit provided buffer and stored −20 °C until slide processing.

CPP_SpyTag synthetic peptides (Pepscan GmbH, Lelystad, The Netherlands, and Mimotopes, Mulgrave, VIC, Australia; sequences in Table A1) were conjugated to SpyC_BLA proteins at a peptide:protein ratio of 1.125:1 or 1.25:1, with a 50 µM final concentration for the SpyC_BLA protein. Reactions were performed in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 200 mM NaCl (pH 7.0) and incubated for 2 h at room temperature with gentle mixing and/or or left overnight at 4 °C. Conjugation efficiencies were analyzed on 4–12% NuPAGE Bis-Tris protein gels stained with SimplyBlue^TM Safe Stain (Invitrogen).

Venoms were milked from V. aspis aspis or V. berus berus snakes harvested in Northern Italy. Antivenom#1 and Antivenom#2 were provided by the Poison Control Centre and National Toxicology Information Centre of Pavia, ICS Hospital. SimplyBlue^TM SafeStain is from Invitrogen. Cytosine β-D-arabinofuranoside hydrochloride (C6645), DNAse I from bovine pancreas (DN25), poly-L-lysine hydrobromide (P1274), Trypsin (T4799) and β-BTx are from Sigma Aldrich. µ-Conotoxin GIIIB is from Alomone, Jerusalem, Israel. Protease inhibitors cocktail is from Roche. NuPage 12% Bis-Tris gels and MES buffer are from Life technologies. Protran nitrocellulose membranes is from Whatman. Luminata^TM is from Merck Millipore. Primary antibodies: anti syntaxin-1A1B polyclonal antibodies have been produced in our laboratory. Secondary antibodies for western blotting are from Calbiochem^®; secondary antibodies for immunofluorescence and α-bungarotoxin-Alexa 555 are from Thermo Scientific, Waltham, MA, USA.

Purified proteins were separated by SDS-PAGE, transferred to PVDF membranes which were probed with Penta-His mouse monoclonal antibody (Qiagen, Hilden, Germany) or a rabbit anti-OVGP1 polyclonal antibody (Abcam, Cambridge, Great Britain) prior to visualization by chemiluminescence. Proteins were treated with N-glycosidase F (Roche^®, Mannheim, Germany) before separation on SDS-PAGE. Proteins were also stained with Simply Blue^TM Safe Stain (Invitrogen, Carlsbad, CA) after SDS-PAGE.
IVM porcine oocytes were incubated for 1 hr at 37 °C in 125 μg/mL of pOVGP1, pOVGP1ab and rOVGP1, lysed and analyzed by immunoblot with a rabbit anti-OVGP1 polyclonal antibody. β-actin was used as a loading control. Average data from three experiments were quantified by image analysis.