Recharging Ni Resin

''Ni resin can be used for up to three or four purifications of the same protein without recharging. To do so, wash the resin with 0.5 M NaOH for 30 minutes and equilibrate the resin with the appropriate binding buffer. If you need to recharge the resin, follow the protocol below.''

'' It is not recommended to recharge the resin more than three times. ''

Recharged resin should be reused for purification of the same recombinant protein.

Materials

 * Used Ni resin
 * EDTA (50 mM)
 * NaOH (0.5 M)
 * NiCl2•6H2O (5 mg/mL)
 * Sodium Azide (0.02%)

Method (recharges 1 mL of resin)

 * 1) Begin by pooling together resin used for the same recombinant protein in a single 15 ml conical tube.
 * 2) Wash the Ni resin twice with 4 mL of 50 mM EDTA to strip away the chelated nickel ions.
 * 3) Wash the Ni resin twice with 4 mL of 0.5 M NaOH.
 * 4) Wash the Ni resin twice with 4 mL of ddH2O.
 * 5) Recharge the column with two washes of 4 mL NiCl2•6H2O prepared in ddH2O.
 * 6) Wash the resin twice with 4 mL of ddH2O.
 * 7) Add 0.02% sodium azide or 20% ethanol as a preservative and cap the tube. Store at 4 C.