Feb 02, 2024
Formation and isolation of Clu phospholipid particles
- Patricia Yuste-Checa1,
- Andreas Bracher1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Patricia Yuste-Checa, Andreas Bracher, F Ulrich Hartl 2024. Formation and isolation of Clu phospholipid particles. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6x59zlqe/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: January 26, 2024
Last Modified: June 01, 2024
Protocol Integer ID: 94600
Keywords: ASAPCRN, isolation of clu phospholipid, clu phospholipid, phospholipid particle, using purified clusterin, purified clusterin from hek293e cell, phosphocholine, clusterin, hek293e cell
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently make in vitro and isolate Clu-phospholipid particles using purified Clusterin from HEK293E cells (dx.doi.org/10.17504/protocols.io.bvvkn64w) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).
Attachments
976-2533.docx
1,020KB
Materials
NativePAGE 3%–12% Bis-Tris SDS gel Thermo Fisher ScientificCatalog #BN1001BOX
NativePAGE™ Sample Buffer (4X)Thermo FisherCatalog #BN2003
NativePAGE™ Running Buffer (20X)Thermo FisherCatalog #BN2001
InstantBlue® Coomassie Protein Stain (ISB1L) (ab119211)AbcamCatalog #119211
Equipment
Glass vial
NAME
Waters Corporation
BRAND
186000272C
SKU
LINK
Troubleshooting
Formation of Clu-phospholipid particles
17h 30m
Prepare 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) stock solution: 5-25 mg/ml DMPC in 3:1 Chloroform:Methanol and store it at -80 °C .
Transfer the corresponding amount of DMPC solution to a glass vial and remove the solvent by evaporation through a constant stream of nitrogen gas until it dries out and becomes waxy.
Note
Avoid overdrying the lipids (extensive white film). If it happens, the lipids can be dissolved in 3:1 Chloroform:Methanol and dried again.
Resuspend the lipids with 1x PBS 7.2 to obtain the desired concentration, vortex and sonicate in a Bioruptor sonication bath (Diagenode) (25 cycles of 5 seconds on – 5 seconds off), or similar. The resulting mixture is turbid and white.
Mix 10 micromolar (µM) Clusterin with 10 millimolar (mM) DMPC in a PCR tube for a Clusterin:DMPC ratio 1:1000. For example, 10 µL Clusterin 20 micromolar (µM) + 10 µL DMPC 20 millimolar (mM) in PCR tubes.
Note
1:1000 Clusterin:DMPC ratio results in extensive Clusterin lipidation. The ratio can be increased and the reaction can be scaled up to obtain high amounts of Clu-phospholipid particles, e.g. for further isolation by size exclusion chromatography.
Incubate the sample through 3 cycles of 18 °C for 15 minutes – 30 °C for 15 minutes using a PCR thermocycler.
1h 30m
Incubate the sample at 18 °C for 00:15:00 – 30 °C for 00:15:00 using a PCR thermocycler (1/3).
30m
Incubate the sample at 18 °C for 00:15:00 – 30 °C for 00:15:00 using a PCR thermocycler (2/3).
30m
Incubate the sample at 18 °C for 00:15:00 – 30 °C for 00:15:00 using a PCR thermocycler (3/3).
30m
Analyze Clusterin lipidation by Native polyacrylamide gel electrophoresis (Native-PAGE). Mix the
samples with NativePAGE Sample Buffer (4X) (refer materials section), load them on a NativePAGE 3%–12% Bis-Tris SDS gel and run the gel in NativePAGE running buffer (refer materials section) at 140 V.
Note
Analysis of protein staining (Coomassie) and lipid staining (Sudan black B) should be done in
independent gels.
For protein staining, incubate the gel Overnight with InstantBlue and de-stain next day with water.
8h
For lipid staining, incubate the gel Overnight with 0.4% Sudan black B (MERCK, S0395) in 16.7%
acetone, 12.5% acetic acid solution (previously centrifuged to remove precipitates) and de-stain the next day with 20% acetone, 15% acetic acid.
8h
Isolation of Clu-phospholipid nanodisc complexes
Centrifuge lipidated Clusterin using a table top centrifuge for 30 seconds to pellet big multi-lamellar lipid vesicles (white pellet).
Load the supernatant into a Superose 6 previously equilibrated with 1x PBS. Clu-phospholipid
nanodisc complexes elute in the first fractions after void volume.
Collect and concentrate the Clu-phospholipid complex containing fractions by ultrafiltration using
Vivaspin MWCO 10.000 (GE Healthcare).
Note
Even after isolation of Clu-phospholipid particles, some free Clusterin impurity is observed likely due to a dynamic exchange between free and lipidated Clusterin.
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.