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) 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 …
Disclosures
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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Cite This Article
Calcium-Dependent Hydrophobic Interaction Chromatography: A Technique to Purify Calcium-Binding Proteins Based on Hydrophobic Interactions. J. Vis. Exp. (Pending Publication), e21092, doi: (2023).