4.2. Venom Fractionation by Reverse Phase Chromatography

Individual B. atrox venoms were fractionated using reversed-phase high-performance liquid chromatography (RP-HPLC) following previously described methods [4]. Briefly, 5 mg of crude lyophilized venom was dissolved in 250 μL of 0.1% trifluoroacetic acid (TFA) and injected onto a Vydac C18 column (250 mm × 4.6 mm, 10 µm particle size) coupled to a Shimadzu LC 20-AT HPLC system. Proteins were eluted at 2 mL/min with a gradient of 0.1% TFA in water (solution A) and acetonitrile (solution B) (5% B for 5 min, 5–15% B over 10 min, 15–45% B over 60 min, 45–70% B over 10 min, 70–100% B over 5 min, and 100% B for 10 min). The fractionation was monitored at 214 nm. The venom profiles of samples collected at the first venom extraction (TI) and samples extracted in periods longer than 2 years were compared individually, with respect to retention times and the height of eluted peaks. Variable fractions had their composition predicted according to peak shape and retention of a previous comprehensive proteomic analysis of B. atrox venom from snakes collected in the same areas in which the major HPLC chromatographic peaks had their components identified by mass spectrometry [50] and corresponded to the elution profiles of the major venom protein families described from venoms of viper snakes [47].