Anion-Exchange Chromatography-Based Protein Purification: A Separation Technique to Isolate a Protein of Interest From Dialyzed Bacterial Lysate Based on Net Charge
Published: April 30, 2023
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 Anion-exchange chromatography Dialysis 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 vo…
Representative 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 …
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The authors have nothing to disclose.
