Crotalus atrox venom

Bovine cardiolipin (1,3-diphosphatidyl-sn-glycerol); dioleoylphosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC); and dioleoylphosphatidylethanolamine (l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, DOPE), each in chloroform, were obtained from Avanti Polar Lipids, Inc. Dodecyl maltoside (DM) was obtained from Anatrace. Horse heart cytochrome c (type III); Crotalus atrox venom; N,N,N',N'-tetramethyl-1,4-benzenediamine dihydrochloride (TMPD); carbonyl cyanide 3-chlorophenylhydrazone (CCCP); valinomycin; phenol red; sodium ascorbate; and sodium cholate were obtained from Sigma Chemical Co. Bio-Beads SM-2 were purchased from Bio-Rad Laboratories. All other chemicals were reagent grade.

Crotalus atrox venom (V7000, Sigma-Aldrich) was reconstituted in 0.065% TFA and 2% acetonitrile (Buffer A) to a concentration of 6 mg/mL. This solution was centrifuged for 10 min at 15,000 g to remove undissolved particles. The sample (100 μL) was fractionated using an AKTAmicro HPLC system (GE Healthcare Life Sciences, Piscataway, NJ, USA) fitted with two reversed-phase columns (SOURCE 5RPC ST polystyrene/divinyl benzene, 4.6×150 mm; GE Healthcare) run in series (flow rate=0.5 mL/min, linear gradient of 0–100% Buffer B (0.05% TFA, 80% acetonitrile) in 30 column volumes). Protein elution was monitored at 214 nm and the peaks were collected manually. Each peak was sent for LC-MS analysis. Chromatography fractionation was performed several more times and we combined several eluted peaks between 62 mL and 68 mL into what we term Fraction 2 (Figure 1). The combined peaks were dried in a speed-vac and then reconstituted in normal saline before injection into the animals.