Mar 27, 2025

Public workspaceClusterin Phospholipid Particles Flotation Assay Using an Optiprep Step Gradient

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vz6mq4gx1/v1
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Patricia Yuste-Checa
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vz6mq4gx1/v1
Protocol Citation: Patricia Yuste-Checa, F Ulrich Hartl 2025. Clusterin Phospholipid Particles Flotation Assay Using an Optiprep Step Gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz6mq4gx1/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 20, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 124798
Keywords: ASAPCRN, clusterin phospholipid particles flotation assay, separate clusterin phospholipid particle, free clusterin, clusterin, optiprep step gradient this protocol, optiprep step density gradient, optiprep step gradient, binders by flotation, flotation, dmpc
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol is based on Yeh FL et al. Neuron (2016) and details how to efficiently separate Clusterin phospholipid particles (Clusterin:DMPC) from free Clusterin together with binders by flotation using an Optiprep step density gradient.
Materials
Reagents
  • PBS
  • 2% Na-deoxycholate
  • Trichloroacetic acid
  • acetone
  • OptiPrep Density Gradient Medium (Merck, D1556)
  • 3.5 mL Thickwall Polycarbonate Tube (349622, Beckman Coulter)
  • NuPAGE™ LDS Sample Buffer (4X) buffer (Thermo Fisher Scientific)

Equipments
SW55 Ti rotor (Beckman Coulter)
Bioruptor sonication bath (Diagenode)

ReagentOptiPrep™ Density Gradient MediumMerck MilliporeSigma (Sigma-Aldrich)Catalog #D1556)
Reagent3.5 mL, Open-Top Thickwall Polycarbonate Tube, 13 x 51mm - 25PkBeckman CoulterCatalog #349622



Troubleshooting
Rhodanese Aggregation Assay
Perform the rhodanese aggregation reaction* in the presence of Clusterin or purified Clu:DMPC nanodiscs** at molar ratio D-Rhod:Clu 1:3.

Note
*See protocol 'Prevention of rhodanese aggregation assay'.
** See 'Formation and isolation of Clu-phospholipid particles'.  dx.doi.org/10.17504/protocols.io.bp2l6x59zlqe/v1

Centrifuge the rhodanese aggregation reactions in the presence of Clusterin or Clu:DMPC at Centrifigation20000 x g, 4°C, 00:15:00 to remove big aggregates.

Note
NOTE: Here we use D-Rhodanese, but binding of other Clusterin substrates to Clusterin:DMPC nanodiscs can be tested and isolated using this assay.


15m
Centrifigation
Optiprep Gradient
Mix the supernatant with OptiPrep Density Gradient Medium (Merck, D1556, 60% iodixanol) to a final concentration of 36% iodixanol in PBS and final volume of Amount1 mL in a 3.5 mL Thickwall Polycarbonate Tube (349622, Beckman Coulter).
Mix
Carefully place Amount1 mL of 24% iodixanol diluted in PBS on top of the 36% iodixanol layer.

Carefully place Amount1 mL of PBS as top layer.

Centrifuge the gradient at Centrifigation54000 rpm, 4°C, 03:00:00 using a SW55 Ti rotor (Beckman Coulter).

3h
Centrifigation
After centrifugation, carefully collect 6 fractions of Amount0.5 mL .

TCA Precipitation for SDS-PAGE Analysis
Dilute each fraction with Amount0.5 mL PBS.

Add Amount40 µL of 2% Na-deoxycholate and vortex.

Pipetting
Incubate TemperatureOn ice for Duration00:15:00 .

15m
Incubation
Add Amount100 µL of 100% Trichloroacetic acid and vortex.

Pipetting
Incubate TemperatureOn ice for Duration01:00:00 .

1h
Incubation
Centrifuge the samples at Centrifigation20000 x g, 4°C, 00:30:00 .

30m
Centrifigation
Add Amount500 µL of ice-cold acetone.
Pipetting
Vortex or sonicate in a Bioruptor sonication bath (Diagenode) (2 cycles of Duration00:00:30 on – Duration00:00:30 off) for faster resuspension.
Centrifuge at Centrifigation200000 x g, 4°C, 00:10:00 .

10m
Centrifigation
Air dry the pellets.
Add Amount30 µL Concentration300 millimolar (mM) Tris Ph8.8 .

Pipetting
Incubate TemperatureOn ice for Duration00:05:00 .

5m
Incubation
Add Amount30 µL NuPAGE™ LDS Sample Buffer (4X) buffer (Thermo Fisher Scientific) containing Concentration100 millimolar (mM) DTT and boil the samples for Duration00:10:00 .

10m
Pipetting
Separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting.

Note
NOTE: Isolated Clusterin phospholipid particles bound to a substrate can be also used for further experiments like cellular uptake, see Yeh FL et al. Neuron (2016).

Protocol references
Yeh FL, Wang Y, Tom I, Gonzalez LC, Sheng M. TREM2 Binds to Apolipoproteins, Including APOE and CLU/APOJ, and Thereby Facilitates Uptake of Amyloid-Beta by Microglia. Neuron. 2016 Jul 20;91(2):328-40. doi: 10.1016/j.neuron.2016.06.015. PMID: 27477018.