Protocol mammalian His-tag purification, written by Rosa Schier

Wash buffer: 500mM NaCl + 50mM Hepes pH 7.5, add up to 1l with ddH2O and filter 0.22um

Elution buffer: 500mM NaCl + 50mM Hepes pH 7.5 + 500mM imidazole pH7.5, add up to 1l with ddH2O and filter 0.22um

 Day before the purification:

• Take a sample of the supernatant for the gel
• Put the beads (10ml/L) (Ni Advance Resin from ProteinArk #Fastback-NiAdv) in the supernatant and let it shake slowly overnight

Day of the purification:

• Let the beads settle
• Get the maximum of FT without the beads in a bottle
• Get the rest of the FT with the column (beads stay in the column) + gel sample
• Every Wash and Elution is collected in a different falcon/bottle + on ice (4°C)
• Wash with 20 CV Wash buffer + gel sample
• Elution 1 with 10 CV = 25mM imidazole final (Elut buffer: 500mM X volume you need to pipet =25mM x final volume) + adjust with Wash buffer + gel sample
• Elution 2 with 10 CV = 50mM imidazole final (Elut buffer: CiVi=CfVf) + adjust with Wash buffer + gel sample
• Elution 3 with 10 CV = 250mM imidazole final (Elut buffer: CiVi=CfVf) + adjust with Wash buffer + gel sample
• Elution 4 with 10 CV of Elut buffer (500mM imidazole final) + gel sample
• Run a gel to see witch fraction to keep
• Do a dialysis with the fractions where we have protein
• Take out of dialysis, measurer the concentration A280nm on the nanodrop and concentrate if necessary