Overview
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 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).
- 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.
- 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. - 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.
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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 |
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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 |
