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Purification of 6x His-tagged Protein (from E. coli)

Bio-protocol Editor Bio-protocol Editor

Published: Jan 5, 2011 DOI: 10.21769/BioProtoc.8 Views: 27694

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Abstract

A polyhistidine-tag is an amino acid motif that contains at least six histidine (His) residues, usually at the N- or C-terminus of the protein. This tag can also be referred to as a hexa histidine-tag or a 6x His-tag. The protocol described here has been developed to purify His-tagged proteins from E. coli under denaturing conditions using Ni-NTA agarose beads.

Materials and Reagents

  1. Ni-NTA superflow (QIAGEN)
  2. Tris base
  3. Urea
  4. IPTG
  5. NaH2PO4·H2O
  6. NaOH
  7. HCl
  8. LB/Amp media
  9. Buffer B (see Recipes)
  10. Buffer C (see Recipes)
  11. Buffer D (see Recipes)
  12. Buffer E (see Recipes)

Equipment

  1. Centrifuge and rotor JLA8.1000 and JA-20 (Beckman Coulter)
  2. Sonicator

Procedure

Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.

Category

Biochemistry > Protein > Isolation and purification

Microbiology > Microbial biochemistry > Protein

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1 Q&A

The concentration of IPTG specified for induction is approximately...
edit 1 Answer 1 View Mar 6, 2014