Biologic biofrac fraction collector

Venom components were separated using anion-exchange chromatography followed by size exclusion chromatography. For anion-exchange chromatography, a similar process was applied as described previously ^{15 (link)}. Briefly, venom proteins from ~1000 venom reservoirs were loaded on Bio SAX column (5 μm, 4.6 × 50 mm, Agilent, USA) using a Biologic Duo-Flow high-performance chromatography system (Bio-rad, Germany). Proteins were eluted at a flow rate of 0.5 ml/min with a gradient of buffer B (25 mM Tris-HCl, 1 M NaCl, pH = 7.5) against buffer A (25 mM Tris-HCl, pH = 7.5). Protein level was monitored by absorbance at 280 nm. Fractions of 200 μl were collected in a deep well plate (Eppendorf, Germany) using the BioLogic BioFrac fraction collector (Bio-rad, Germany), then desalted using Zeba spin desalting plates (Thermo Scientific, USA). Fractions 56 and 57, which showed the highest cytotoxicity on Hi-5 cells, were pooled for further size exclusion chromatography using Agilent Bio SEC columns (3 μm, 300 A, 4.6 × 300 mm) at a flow rate of 0.2 ml/min using PBS as the running buffer. Protein level was monitored by absorbance at 214 nm. The fraction with cytotoxicity was pooled and subjected to SDS-PAGE.