Ibright cl1000 gel documentation system

0.05 µg/µl of crude venom in 1X phosphate buffered saline (PBS) was mixed with purified calf thymus DNA (Sigma-Aldrich, United States) and incubated at 37°C for 60 min to assess the DNase activity of N. sagittifera and N. naja venoms (Gerceker et al., 2009 (link); Senji Laxme R. R. et al., 2021 (link)). Post-incubation, these reaction mixtures were subjected to 0.8% agarose gel electrophoresis. GelRed (Sigma-Aldrich, United States) nucleic acid stain was used to visualise DNA in an iBright CL1000 gel documentation system (Thermo Fisher Scientific, United States). The percentage DNase activity of the venom samples, and that of the positive control, was calculated by densitometric analysis of the images using the ImageJ software (Schneider et al., 2012 (link))

The fibrinogenolytic activity of N. sagittifera and N. naja venoms was elucidated using an electrophoresis-based protocol (Teng et al., 1985 (link)). A predefined concentration of venom (1.5 µg) based on protein content was incubated with human fibrinogen (15 µg) (Sigma-Aldrich, United States) in 1X PBS (pH 7.4) at 37°C for 60 min. An equal volume of the loading dye solution (1 M Tris-HCl, pH 6.8; 50% Glycerol; 0.5% Bromophenol blue; 10% SDS; and 20% β-mercaptoethanol) was added to the reaction mixture, and the resulting mixture was then heated at 70°C for 10 min. Samples were subjected to 15% SDS-PAGE, and the gel was subsequently stained with Coomassie Brilliant Blue R-250. An iBright CL1000 gel documentation system (Thermo Fisher Scientific, United States) was used to visualise the stained gels. A negative control containing human fibrinogen alone was used to compare and interpret the results.