Fractionation

Cation Exchange Chromatography

  • A Beckman Capillary electrophoresis P/ACE 5500 is used to observe the purity of venom fractions.

  • Ten microliters of 1 mg/mL fractions are mixed with 20 µl of 0.01M Borate buffer, pH 8.3.

  • The mixture is immediately separated for 10 min at 20 kV, 19.5 µamps, using a 0.01M Borate buffer, pH 8.3 through a 75 µm I.D. X 50 cm (100 x 800 aperture) free zone capillary.

  • A P/ACE UV absorbance detector at 214 nm is used to detect the peptides.

Anion Exchange Chromatography

  • Each venom sample is fractionated by anion exchange chromatography.

  • Crude venom (30 mg) is injected into a Waters Protein-Pak DEAE 5PW (7.5 x 75 mm) HPLC column.

  • Fractions are eluted using a 0.02 M Tris-HCl (tris (hydroxymethyl) aminomethane-hydrochloric acid) buffer, pH 8.0, and 0.5 M NaCl (sodium chloride) gradient over 60 min with a flow rate of 1 ml/min.

  • A Waters 484 tunable detector is used to monitor absorbances at 280 nm.

  • Millennium software v.4.0 is used to control the pumps and store the data.

  • Fractions are stored at -90˚C. To perform the proteolytic assays, the fractions are dialyzed against Milli-Q water for 8 hr, lyophilized, and reconstituted in 0.01 M phosphate buffered saline (PBS), pH 7.4, 0.15 M NaCl.

  • Protein concentrations are determined by standard methods at 280 nm using BSA.

Reverse Phase Chromatography

  • Venom is fractionated by reverse phase chromatography.

  • Crude venom (5 mg) is injected into a Grace Vydac Reverse Phase C18 (4.6 x 150 mm) column.

  • Fractions are eluted using a 0.1% TFA, and 80% Acetonitrile in 0.1%TFA gradient over 60 min with a flow rate of 1 mL/min.

  • A Waters 484 tunable detector is used to monitor absorbances at 280 nm.

  • Millennium software v.4.0 is used to control the pumps and store the data.

  • Fractions are stored at -90˚C. To perform the proteolytic assays, the fractions are lyophilized, and reconstituted in 0.01 M phosphate buffered saline (PBS), pH 7.4, 0.15 M NaCl.

  • Protein concentrations are determined by standard BSA methods at 280 nm.

Gel Filtration Chromatography

  • A total of 50 µL of crude venoms or fractions (total protein depends on sample availability) are separated by size exclusion chromatography using a Waters Protein-Pak60 (1,000-20,000 Da separating ranges), 125 (2,000-80,000 Da) or 300 (10,000-400,000 Da) column.

  • A 0.02M Sodium Phosphate buffer, pH 6.5 and a flow rate of 0.5 ml/min are be used to elute the proteins.

  • Acquisition and storage of data and pump control is be accomplished by the Waters™ HPLC system and Millennium Software v.4.0.

Dialysis and Concentration of Proteins

  • Venom fractions collected by cation exchange chromatography are dialyzed against Milli-Q water for 12 hr using Spectra/Por® dialyzing membrane (several molecular weight cut offs are available) and concentrated by lyophilization under a 15 micron vacuum at –40˚C using a LABCONCO Freeze Dryer 4.5