NEBExpress® Ni Resin Quick Start Protocol (NEB #S1428)
Lysis/Binding Buffer: 20 mM sodium phosphate, 300 mM NaCl, pH 7.4
Wash Buffer: 20 mM sodium phosphate, 300 mM NaCl, 5 mM Imidazole, pH 7.4
Elution Buffer: 20 mM sodium phosphate, 300 mM NaCl, 500mM Imidazole, pH 7.4
- Load resin into a gravity flow column and equilibrate with ≥ 5 column volumes (CV) of Lysis/Binding buffer.
- Load His-tagged protein lysate with a slow flow rate at 4°C, allow interaction with the resin for ≥ 5 minutes.
- Collect the flow through in a clean tube.
- Wash column with ≥ 10 CV of Wash buffer.
- Elute fusion protein with Elution buffer and collect flow through in fractions.
- Monitor protein elution by measuring the absorbance at 280 nm or by SDS-PAGE.
- Analyze the protein lysate (load), flow through, washes and eluates by SDS-PAGE.