NEBExpress^® Ni Resin Quick Start Protocol (NEB #S1428)

Column Buffers:

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

1. Load resin into a gravity flow column and equilibrate with ≥ 5 column volumes (CV) of Lysis/Binding buffer.
   
   
2. Load His-tagged protein lysate with a slow flow rate at 4°C, allow interaction with the resin for ≥ 5 minutes.
   
   
3. Collect the flow through in a clean tube.
   
   
4. Wash column with ≥ 10 CV of Wash buffer.
   
   
5. Elute fusion protein with Elution buffer and collect flow through in fractions.
   
   
6. Monitor protein elution by measuring the absorbance at 280 nm or by SDS-PAGE. 
   
   
7. Analyze the protein lysate (load), flow through, washes and eluates by SDS-PAGE.