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

Protocol

1. Calcium-dependent hydrophobic-interaction chromatography (HIC)

1. Dialysis
   1. Dialyze the protein solution against 20 mM Tris, 140 mM NaCl, pH 7.5
2. Chromatography
   1. 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 to the sample to a final concentration of 25 mM and filter through 0.45 µm. Equilibrate HIC buffers and sample to 4 °C (column temperature).
   2. Start the liquid chromatography system with general maintenance, connect column buffers HIC A and B and the column. Refer to Table 1 for further chromatographic parameters.
   3. Equilibrate the column, load the sample and extend the ‘wash unbound sample’ block until the UV signal reaches the baseline level again. Then start elution with a calcium chelator containing buffer (EDTA). Refer to Table 2 for a detailed method protocol.
      NOTE: Previous experiments have shown that an excess of calcium seems to be beneficial for binding of S100A12 to the chromatography resin.
   4. Collect peak fractions of 2 mL and analyze 10 µL of each fraction on a Coomassie-stained 15% SDS-PAGE. Pool pure S100A12 fractions and dialyze against Hepes-buffered saline (HBS; 20 mM Hepes, 140 mM NaCl, pH 7.0).
      NOTE: Extinction coefficient of monomeric S100A12 is 2980 M-1 cm-1.

Access restricted. Please log in or start a trial to view this content.

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	n/a	A	Waste
Wash out unbound sample	1−2 CVs	A	High volume outlet
Elution	100 % Buffer B	B	Fraction collector
Wash out–Buffer B	1 CV	B	Waste
Re-Equilibration	2 CVs	A	Waste

Table 2: Detailed information on the used method of hydrophobic-interaction chromatography.

Bed Volume (CV)	320 mL
Monitor	Absorbance at 280 nm
Pressure Max	3 bar
Column buffer A	20 mM Hepes, 140 mM NaCl, pH 7.2
Sample Volume	Up to 13 mL
Flow Rate	1−1.5 mL/min
Temperature	12−15 °C

Access restricted. Please log in or start a trial to view this content.

Materials

Name	Company	Catalog Number	Comments
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