Superose 12 hr 10 30 column

Venom was fractioned by molecular exclusion chromatography on a Superose 12 HR 10/30 column (Amersham Pharmacia Biotech AB, Uppsala, Sweden). All peak profiles were monitored by their absorbance at 280 nm using a UPC-900 monitor (Amersham Pharmacia Biotech AB). Briefly, in a climate-controlled room (22 ± 2 °C), 20 milligrams of venom was dissolved in five milliliters of column eluent and 500 μL was applied each time into the column, previously equilibrated with ammonium acetate 50 mM. In the same eluent, the proteins were eluted at a flow rate of 0.4 mL/min, and fractions were manually collected. Fraction 3 (3 mg/mL), obtained from gel filtration chromatography, was pooled and submitted to another cycle of molecular exclusion using a Superdex 75 10/300 GL column (GE Healthcare, Bio-Sciences AB, Uppsala, Sweden), following the abovementioned conditions. Proteins were freeze-dried, resuspended in sterile PBS and stored at − 20 °C. The protein content of the obtained fractions was estimated by BCA assay and the electrophoretic profile was visualized by SDS-PAGE [41 (link)] (4.0 μg/well resolved in 10% polyacrylamide gel) and silver-stained [42 (link)].

One milligram (1 mg) of commercial horse F(ab’)₂ anti-botulinum AB (bivalent), anti-diphteric, antitetanic or anti-rabies immunoglobulins were subjected to molecular exclusion chromatography on a Superose 12 HR 10/30 column (Amersham Pharmacia Biotech AB, Sweden), equilibrated and eluted with ammonium acetate 50 mM, pH 7.4. Samples were run at a 24 mL/h flow rate, and their protein content was monitored by recording the absorbance at 280 nm in a UPC-900 Amersham Pharmacia Biotech.