Mar 19, 2025
Micro-scale Analytical Size Exclusion Chromatography of Clusterin
- Andreas Bracher1,
- Patricia Yuste-Checa1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry, Martinsried, Germany
Protocol Citation: Andreas Bracher, Patricia Yuste-Checa, F Ulrich Hartl 2025. Micro-scale Analytical Size Exclusion Chromatography of Clusterin. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4wm4ovo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 11, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 124317
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to analyze pH-dependent changes in the oligomeric state of Clusterin by analytical size exclusion chromatography (SEC).
Materials
Buffers and reagents:
- Buffer A:
| A | B | |
| Na-acetate pH 5.0 | 20 mM | |
| NaCl | 100 mM | |
| Ethylenediaminetetraacetic acid (EDTA) | 1 mM |
- Buffer B:
| A | B | |
| 2-(N-morpholino)ethanesulfonic acid (MES)-NaOH pH 6.5 | 20 mM | |
| NaCl | 100 mM | |
| EDTA | 1 mM |
- Buffer C:
| A | B | |
| 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH pH 7.5 | 20 mM | |
| NaCl | 100 mM | |
| EDTA | 1 mM |
- Buffer D:
| A | B | |
| Tris(hydroxymethyl)aminomethane (Tris)-HCl pH 7.5 | 20 mM | |
| NaCl | 100 mM | |
| EDTA | 1 mM |
Note
Prepare buffers A-D by mixing and diluting the following stock solutions with Milli-Q (Merck) de-ionized water: 1 M Na-acetate pH 5.0, 1 M MES-NaOH pH 6.5, 1 M HEPES-NaOH pH 7.5, 1 M Tris-HCl pH 8.5, 5 M NaCl, and 0.5 M EDTA-NaOH pH 8.0. Preparation: The first three stocks are prepared by titrating 1 mol acetic acid, 1 mol MES free acid and 1 mol HEPES free acid, respectively, each dissolved in 0.7 l Milli-Q water with 5 M NaOH to the respective pH value (NaOH is corrosive: Wear protective goggles and gloves. Heat development: Let cool to room temperature before final pH adjustment), followed by topping up with Milli-Q water to 1.00 l total volume. 1 mol Trizma base (Tris, Merck) dissolved in 0.7 l Milli-Q water is titrated to pH 8.5 with fuming hydrochloric acid (HCl) (HCl is corrosive: Wear protective goggles and gloves. Heat development: Let cool to room temperature before final pH adjustment), followed by topping up with Milli-Q water to 1.00 l total volume. EDTA free acid (0.25 mol) is suspended in 0.35 l Milli-Q water and titrated with 5 M NaOH to pH 8.0 (NaOH is corrosive: Wear protective goggles and gloves. Heat development: Let cool to room temperature before final pH adjustment), while EDTA slowly dissolves. A final volume of 0.50 l is adjusted with Milli-Q water.
Sterile-filter and de-gas buffers A-D in vacuum.
- Clusterin variant (see protocol “Clusterin purification from HEK293E cells”, dx.doi.org/10.17504/protocols.io.bvvkn64w).
Instruments:
- Superdex-200 Increase 3.2/300 column (Cytiva)
- 100 µl-Hamilton syringe, for injection
- 50 µl-loop
- Microscale chromatography system
Note
A NGC chromatography system (Bio-Rad) custom-optimized for small sample volumes was used. Alternatively, a Ettan liquid chromatography system (GE), a Äkta Pure Micro system (Cytiva) or similar can be used as well.
Preparatory Work
Preparatory Work
8h
8h
Dilute the Clusterin variant with the respective buffer to 10 micromolar (µM) concentration. The volume needed for one run is 60 µL sample.
Incubate Overnight or longer On ice (Store ice bucket in cold room for convenience).
8h
Wash the chromatography system with sterile-filtered and de-gassed Milli-Q water (“Purge” or “Pump wash”).
Attach the Superdex-200 column to the chromatography system. Avoid trapping air on the column by filling the connection tubings with Milli-Q water first.
Wash out the storage buffer (20% ethanol) from the Superdex-200 column with 5 mL of Milli-Q water at a flow rate of 0.03 mL min-1. Set the column pressure limit to 1.0 MPa.
Note
The runs are performed at 20 °C in a temperature-controlled room, to avoid problems with high viscosity.
SEC Run
SEC Run
Equilibrate the Superdex-200 column with 5 mL of buffer A at a flow rate of 0.05 mL min-1. Set the column pressure limit to 1.0 MPa.
Note
If the pressure limit is exceeded, replace the pre-column filter.
Program the following method:
0.0 mL Start flow at 0.05 mL min-1, pressure limit 1.0 MPa.
- Monitor absorbance at 280 nm wavelength, conductivity and pressure.
0.2 mL Switch injection valve to “Inject”.
0.25 mL Switch injection valve to “Load”.
0.85 mL Start collecting 100 µL fractions.
2.35 mL Stop fraction collection.
2.7 mL Stop pump flow.
Start the chromatography run. Now you have 00:04:00 for sample injection.
4m
Wash the loop by injecting three times 100 µL buffer A using the Hamilton syringe.
Draw up 60 µL sample into the Hamilton syringe. Avoid drawing air into the syringe.
Inject the liquid content of the Hamilton syringe into the loop. Leave air bubbles in the syringe. Leave the syringe in the fill port.
Note
- Air bubbles in the loop will be compressed by the pre-column pressure and will result in an offset in the elution volumes. During the compression phase the pre-column pressure will drop and then recover.
- Unresolved large aggregates (not observed) will elute as a sharp peak approximately 0.7 ml after injection (the so-called “void volume’). Clusterin dimers and monomers will elute as symmetric peaks of approximately 100 ul half-maximal width at ~1.34 and 1.54 ml elution volume, respectively. Traces of higher Clusterin oligomers might be also visible. Small molecular weight impurities elute at approximately 2.15 ml elution volume (the so-called “salt peak’).
Fig. 1 Example UV-trace (green) for size exclusion chromatography of wildtype Clusterin at 10
µM after equilibration in buffer B.
Note
Because Clusterin is a potent molecular chaperone, it might get trapped on precipitated
protein in the pre-column filters and the column, if the column was used before. Perform
“dummy” runs by injecting Clusterin until the chromatogram UV-trace stabilizes.
For analyzing further Clusterin variants in buffer A, repeat steps 8-11 for the remaining Clusterin variant samples.
For analyzing further buffers, repeat steps 6-12 with the next buffer.
Note
The Superdex-200 column should be size-calibrated with a set of reference proteins of known molecular weight (Thyroglobulin, 670 kDa; γ-globulin, 158 kDa; ovalbumin, 44 kDa; myoglobin, 17 kDa) in PBS buffer. The logarithm of the molecular mass should be proportional to the elution volume.