Purification of Tityus fasciolatus Venom Peptide

Crude venom of Tityus fasciolatus was fractioned by RP-HPLC (Reversed Phase High Performance Liquid Chromatography) (Shimadzu Co., Kyoto, Japan), using a C18 column (Synergi Fusion RP 4 μ, 80 Å, 250 × 4.6 mm (Phenomenex, Inc., Torrance, CA, USA). Components were separated using a linear gradient of solvent A (0.12% TFA in water) and solvent B (0.10% TFA in acetonitrile) from 0 to 60% for 60 min at a 1 mL/min flow rate as described previously [20 (link)]. Three extra steps of RP-HPLC were conducted to purify Tf1a, the first with 0.5%[B]/min, second purification step with 0.5%[B]/min at 45 °C, and the last purification step with 0.3%[B]/min at 45 °C.