Oct 25, 2020

Public workspaceHis-tag purification

DOI
dx.doi.org/10.17504/protocols.io.bnw5mfg6
  • 1University of Groningen
  • iGEM Groningen 2020
a.stan.6
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DOI: dx.doi.org/10.17504/protocols.io.bnw5mfg6
Protocol Citation: Andreea S 2020. His-tag purification . protocols.io https://dx.doi.org/10.17504/protocols.io.bnw5mfg6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Other
The protocol is developed based on literature and has not been tested yet.
Created: October 25, 2020
Last Modified: October 25, 2020
Protocol Integer ID: 43709
Abstract
His tag purification uses the technique of immobilised metal affinity chromatography. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. It has been studied that among amino acids constituting proteins, histidine is strongly involved in the coordinate bond with metal ions. Therefore, if a number of histidines are added to the end of the protein by genetic engineering, the affinity of the protein for the metal ion is remarkably increased and the basic idea is that purification can be easily carried out. When a protein having a His tag is brought into contact with a carrier on which a metal ion such as nickel is immobilized, the histidine residue chelates the metal ion and binds to the carrier. Since other proteins do not bind to the carrier, they can be washed off with a buffer. Thereafter, it is possible to recover the protein having the His tag with high purity.
Separate proteins from soil matrix
Separate proteins from soil matrix
Collect soil samples of Amount5 g

Extract total proteins using NoviPure Soil Protein Kit or other comercially available kits for total protein soil extraction



His-tag separation of NLP
His-tag separation of NLP
1h
1h
Wash the Ni2+-sepharose column material with 12 CVs of MQ and 4 CVs of Column Wash Buffer (Concentration10 millimolar (mM) imidazole, KPi Concentration50 millimolar (mM) Ph7 , NaCl Concentration200 millimolar (mM) ).

Note
Use ±0.5 ml of Ni2+-sepharose column material per 10 mg of total protein.

Apply the sample, add imidazole (10mM final concentration) and the washed Ni2+-sepharose column material. Nutate in at Temperature4 °C for Duration01:00:00

1h
Pour column, collect flow through to apply on SDS gel.
Wash column with 20 CVs of Wash Buffer (Concentration50 millimolar (mM) imidazole, KPi Concentration50 millimolar (mM) Ph7 , NaCl Concentration200 millimolar (mM) ).

Elute protein with Elution Buffer (Concentration500 millimolar (mM) imidazole, Kpi Concentration50 millimolar (mM) Ph7 , NaCl Concentration200 millimolar (mM) ) in Amount200 µL fractions. Check elution fractions Absorbance by NanoDrop.

Run an SDS gel to check purification:
- Soil suspension & Flow through: dilute 15x, applyAmount5 µL
- Wash: dilute 1.25x, apply Amount10 µL
- Elution fractions: dilute to ±0.2 mg/ml, applyAmount5 µL