Kta purifier upc 900

Disintegrin from C. d. collilineatus venom was purified through two chromatographic steps carried out in a Fast Protein Liquid Chromatography (FPLC) system (Äkta Purifier UPC 900, GE Healthcare, Uppsala, Sweden). The venom (30 mg) was dispersed in 0.1% trifluoroacetic acid (TFA, solution A) and 1% formic acid, and centrifuged at 13,000 × g at 4 °C for 10 min. The supernatant was fractionated on a C18 column (250 × 10 mm, 5 μm particles, 300 Å, Phenomenex, Torrence, CA, USA) at a flow rate of 5 mL/min, using the concentration gradient described by Calvete et al. [32 (link)]. The second step was performed on another C18 column (250 × 4.6 mm, 3.6 μm particles, Phenomenex, Torrence, CA, USA) at a flow rate of 1 mL/min and the proteins were eluted using a segmented concentration gradient from 6.3 to 100% of solution B (80% acetonitrile, ACN, in 0.1% TFA). In both steps, the protein elution was monitored by absorbance at 214 nm. Fractions of interest were collected, frozen and lyophilized for further analysis.

The venom was fractionated using a method previously described by Calvete et al. and our group [32 , 33 (link)]. Briefly, the pooled venom (22 mg, 1 mg of each venom) was dispersed in 1.1 mL of 0.1% TFA (solution A) and 1% formic acid, centrifuged at 13,000 × g for 10 min at 4 °C. Fractionation was performed on a C18 column (250 × 10 mm, 5 μm particles, 300 Å, Phenomenex, Torrence, CA, USA) coupled to Fast Protein Liquid Chromatography (FPLC) system (Äkta Purifier UPC 900, GE Healthcare, Uppsala, Sweden). Protein elution was monitored by absorbance at 214 nm and eluted fractions were collected, frozen and lyophilized for further analysis.