Journal: Toxins

Article Title: Insights into the Mechanisms Involved in Strong Hemorrhage and Dermonecrosis Induced by Atroxlysin-Ia, a PI-Class Snake Venom Metalloproteinase

doi: 10.3390/toxins9080239

Figure Lengend Snippet: Purification of the PI-class SVMP from B. atrox venom. Venom samples (50 mg) were applied to HiPrep 16/60 Sephacryl S-200 HR column and eluted at flow rate of 0.5 mL/min in 20 mM Tris/HCl buffer, pH 7.8, containing 150 mM of NaCl ( A ). Fractions containing ~25 kDa proteins presenting hemorrhage and dermonecrosis activities (continuous line under the peaks) were pooled, applied into a Mono Q 5/50 GL anion exchange column and eluted at flow rate of 1 mL/min with a gradient from 0 to 1 M of NaCl ( B ). Pooled fractions (10 µg) indicated in the graphs by continuous line under the peaks were subjected to SDS-PAGE under reducing (R) or non-reducing conditions (NR) and silver-stained. The band indicated by * was excised and submitted to MS/MS.

Article Snippet: Approximately 50 mg of B. atrox venom were dissolved in 200 µL of elution buffer (20 mM Tris-HCl + 150 mM NaCl + 1 mM CaCl 2 , pH 7.8) and centrifuged at 14,000 g for 10 min. After centrifugation, the pellet was discarded and the supernatant was injected into the column HiPrep 16/60 Sephacryl S-200 HR (120 mL 16 × 600 mm, GE Healthcare, Pittsburgh, PA, USA).

Techniques: Purification, SDS Page, Staining, Tandem Mass Spectroscopy