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

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 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)

  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.

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 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

Declarações

The authors have nothing to disclose.

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

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Citar este artigo
Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions. J. Vis. Exp. (Pending Publication), e21092, doi: (2023).

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