Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions

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 the purification of calcium-binding protein from a dialyzed cell lysate through calcium-dependent hydrophobic interaction chromatography. The calcium-binding proteins expose a hydrophobic region upon binding with calcium, facilitating interaction with a hydrophobic group on resin. Later these proteins are eluted using calcium chelator EDTA that reverses the interaction.

Protocol

1. Calcium-dependent hydrophobic-interaction chromatography (HIC) Dialysis Dialyze the protein solution against 20 mM Tris, 140 mM NaCl, pH 7.5 Chromatography Prepare 1 L of chromatography buffer HIC A by dissolving 20 mM Tris, 140 mM NaCl and 25 mM CaCl2 in deionized water and adjust the pH to 7.5. For HIC buffer B, dissolve 20 mM Tris, 140 mM NaCl and 50 mM EDTA. Adjust the pH to 7.0 and filter and degas the buffers. Add CaCl2…

Representative Results

Table 1: Detailed information on the applied parameters of hydrophobic-interaction chromatography. Block Volume Buffer Outlet Equilibration 1−2 column volumes (CVs) A Waste Sample load …

Materials

Chemical
EDTA disodium salt dihydrate	Carl Roth	8043.1
Phenyl Sepharose High Performance	GE Healthcare	17-1082-01	Resin for hydrophobic interaction chromatography
Sodium chloride (NaCl)	Carl Roth	3957.2
Sodium hydroxide	Carl Roth	P031.1
Tris Base	Carl Roth	4855.3
25% HCl	Carl Roth	X897.1
Calciumchlorid Dihydrat	Carl Roth	5239.1
Labware
0,45 µm syringe filter	Merck	SLHA033SS
14 mL roundbottom tubes	BD	352059
2 L Erlenmyer flask	Carl Roth	LY98.1
Fraction collector tubes 5 mL	Greiner	115101
Spectra/Por Dialysis Membrane (3.5 kDa)	Spectrum	132724
Steritop filter unit	Merck	SCGPT01RE
Equipment
Fraction collector	GE Healthcare	Frac-920