Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge

Abstract

Source: Fuehner, S., et al. Purification of Human S100A12 and Its Ion-induced Oligomers for Immune Cell Stimulation J. Vis. Exp. (2019)

In this video, we demonstrate an anion-exchange chromatography technique to purify human calcium-binding protein S100A12 based on the net surface charge of the protein from an Escherichia coli culture.

Protocol

1. Protein Purification

1. Anion-exchange chromatography
   1. Dialysis
      1. Prepare anion-exchange chromatography (AIEX) buffer A by dissolving 20 mM Tris, 1 mM EDTA and 1 mM ethylene glycol-bis(2-aminoethylether)-N, N, N', N'-tetraacetic acid (EGTA) in deionized water and adjust the pH to 8.5 with HCl. For dialysis prepare 2 x5 L and for chromatography 2x 1 L of AIEX buffer A.
         NOTE: The dialysate volume should be at about 100 times the sample volume. All buffers used for chromatography should be filtered (0.45 μm or smaller) and degassed (e.g., by ultrasonic bath or vacuum degassing).

Results

Table 1: Detailed information on the applied parameters of anion-exchange chromatography.

Bed Volume (CV)	75 mL
Monitor	Absorbance at 280 nM
Pressure Max	3 bar
Column buffer A	20 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, pH 8.5
Column buffer .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
25% HCl	Carl Roth	X897.1
4−20% Mini-PROTEAN TGX Protein Gels	BioRad	4561093
Coomassie Briliant Blue R250 Destaining Solution	BioRad	1610438
Coomassie Briliant Blue R250 Staining Solution	BioRad	1610436
EDTA disodium salt dihydrate	Carl Roth	8043.1
EGTA	Carl Roth	3054.3
Q Sepharose Fast Flow	GE Healthcare	17-0510-01	Resin for anion-exchange chromatography
Sodium chloride (NaCl)	Carl Roth	3957.2
Sodium hydroxide	Carl Roth	P031.1
Tris Base	Carl Roth	4855.3
0.45 µm syringe filter	Merck	SLHA033SS
5 L measuring beaker	Carl Roth	CKN3.1
50 mL conical centrifuge tubes	Corning	430829
Dialysis Tubing Closures	Spectrum	132738
Fraction collector tubes 5 mL	Greiner	115101
Spectra/Por Dialysis Membrane (3.5 kDa)	Spectrum	132724
Steritop filter unit	Merck	SCGPT01RE
ÄKTA purifier UPC 10	GE Healthcare		FPLC System
Fraction collector	GE Healthcare	Frac-920
Mini Protean Tetra Cell	BioRad	1658000EDU
NanoPhotometer	Implen	P330
pH meter	Knick	765