Team:Queens Canada/nick

His-Tag Nickel Chromatography · Benchling

His-Tag Nickel Chromatography

Introduction

The Ni-NTA Purification System is designed for the purification of recombinant proteins with the addition of a histidine tag (6 residues). The system is dependent on the affinity and selectivity of Ni-NTA agarose for proteins containing the His-tag. For insoluble proteins for which protein activity is to be preserved, prepare the lysate and column under denaturing conditions, and then use native buffers during the wash and elution steps to allow for refolding of the protein.

Materials

  • Column Preparation:
    • Denaturing Binding Buffer
    • Prepared Ni-NTA Agarose
    • Lysate prepared under denaturing conditions
  • Purification:
    • Lysate
    • Prepared purification column
    • Denaturing Binding Buffer
    • Denaturing Wash Buffer (pH 6.0)
    • Native Wash Buffer
    • Native Elution Buffer

Procedure

  • Preparing Ni-NTA Column (under denaturing conditions)
  1. Resuspend Ni-NTA Agarose by gently inverting the bottle.
  1. Pipet 2mL of resin into a 10-mL purification column, and allow 5-10 minutes for it to settle.
  1. Add 6mL of distilled water and resuspend resion by gently inverting and tapping the column.
  1. Allow resin to settle using gravity for 5-10 minutes.
  1. Add 6mL of denaturing binding buffer and resuspend resin.
  1. Allow resin to settle and gently aspirate the supernatant.
  1. Repeat steps 6-7.
  • Purification (under hybrid conditions)
  1. Add 8mL of lysate to prepared purification column.
  1. Allow binding to occur for 15-30 minutes at room temperature.
  1. Wash the column with 4mL of Denaturing Wash Buffer (pH 6.0).
  1. Wash the column with 8mL Native Wash Buffer. Repeat 3 more times.
  1. Elute the protein with 8-12mL Native Elution Buffer. Collect fractions.