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 2.5×50 cm column.