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Comparative Physiology @ Guelph

WSU Preparation of Affigel Resin

Currently not used in our lab but saved for reference.

Put 100 mL of resin in glass fritted funnel stopped with parafilm to prevent leakage.

  • Add ~100 mL of 10 mM Sodium Acetate pH 5.2 cold. Let resin settle.
  • Pipet off supe, add 200 mL of 10 mM Sodium Acetate pH 5.2 cold. Soak 10 minutes
  • Wash 8x 200 mL 10 mM Sodium Acetate pH 5.2 cold.
  • Prep 6 mg/mL RcTnC in 10 mM Sodium Acetate pH 5.2 cold (600 mg into 100 mL). Stir 30 minutes at 4oC.
  • Split resin into (6) 50 mL conicals. 1:1 dilution of resin:RcTnC, approximately 17.5 mL of each into each concial.
  • Parafilm, rock O/N @ 4oC
  • Next day, spin conicals 2k rpm 2 minutes.
  • Pipet off supe, save for gel.
  • Wash each conical 3x ~25 mL 10 mM Sodium Acetate pH 5.2 cold. Invert to mix. DON’T SHAKE OR VORTEX!!!
  • Prep fresh 6 mg/mL RcTnC in 10 mM Sodium Acetate pH 5.2 cold (600 mg into 100 mL). Stir 30 minutes at 4oC.
  • Add 6 mg/mL RcTnC to conicals in a 1:1 dilution of resin:RcTnC, approximately 17.5 mL of each into each concial.
  • Parafilm, rock O/N @ 4oC
  • Next day, spin conicals 2K rpm 2 minutes.
  • Pipet off supe, save for gel.
  • Wash each conical 3x ~25 mL 10 mM Sodium Acetate pH 5.2 cold. Invert to mix. DON’T SHAKE OR VORTEX!!!
  • Pipet off supe, add same volume of 1 M Ethanolamine-HCl pH 8.0. Rock at RT 1hr 30 min.
  • Spin 2K rpm 2 minutes, pipet off supe.
  • Add same volume of 10 mM Sodium Acetate pH 5.2 cold.
  • Gently invert to mix, pour into cold 600 mL beaker.
  • Wash out each conical 1x with 10 mM Sodium Acetate pH 5.2 cold, add to 600 mL beaker.
  • Add 10 mM Sodium Acetate pH 5.2 cold to 500 mL, allow resin to settle at 4oC.
  • Pipet off supe, add 10 mM Sodium Acetate pH 5.2 cold to 500 mL, allow resin to settle at 4oC.
  • Pipet off super, pour into 2.5x50 cm column.