Jun 11, 2024
Filter trap assay for the detection of alpha-synuclein aggregation
- Cole S Sitron1,
- Victoria A Trinkaus1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Protocol Citation: Cole S Sitron, Victoria A Trinkaus, F Ulrich Hartl 2024. Filter trap assay for the detection of alpha-synuclein aggregation. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v92re4l3e/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 17, 2024
Last Modified: June 11, 2024
Protocol Integer ID: 101394
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol describes an assay that detects aggregated alpha-synuclein in cell lysates.
Materials
Cell culture
Cells with alpha-synuclein aggregates, induced as in [dx.doi.org/10.17504/protocols.io.eq2lyjbbplx9/v1], growing in 12-well plates.
Reagents
- TrypLE™ ExpressThermo Fisher ScientificCatalog #12605010
- Pierce™ Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225
Buffers
- 10% FBS (Fetal Bovine SerumGibco - Thermo FischerCatalog #10270106 ) in 1X PBS (diluted from PBS (10X), pH 7.4Thermo Fisher ScientificCatalog #70011051 )
- 1X PBS
- Lysis Buffer:
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| NaCl | 150 mM | |
| Triton-x100 | 1% (v/v) | |
| cOmplete protease inhibitor tablet | 1 mini tablet per 15 ml buffer (Sigma-Aldrich cat. no. 4693159001) | |
| Phos-STOP phosphatase inhibitor tablet | 1 tablet per 15 ml buffer (Sigma-Aldrich cat. no. 4906845001) | |
| Benzonase ((Max Planck Institute of Biochemistry Core Facility) | 7.5 U/ml |
- Wash Buffer:
| A | B | |
| Tris-HCl pH 7.5 | 50 mM | |
| NaCl | 150 mM | |
| Triton-x100 | 1% (v/v) |
Consumables
- 0.2 micrometer pore size cellulose acetate membrane (e.g. GE cat. no. 10404131)
- PARAFILM® MMerck MilliporeSigma (Sigma-Aldrich)Catalog #P7793
Equipment
Equipment
Bioruptor® Plus sonication device
NAME
Sonication device
TYPE
Bioruptor®
BRAND
B01020001
SKU
LINK
- Slot blot vacuum manifold (e.g. Hoefer PR648)
- Fume hood
Harvesting cells
Harvesting cells
Prepare lysis buffer and place On ice .
After 24:00:00 of exposure to PFFs/PBS, wash wells 1X with 1 mL warm PBS.
1d
Withdraw PBS and treat wells with 300 µL of TrypLE.
Quench dissociation reagent with 300 µL of 10% FBS and transfer the contents of the wells to 1.5 ml tubes.
Centrifuge 1500 x g for 00:03:00 .
3m
Withdraw the supernatant and wash cells with 1 mL PBS.
Centrifuge 1500 x g for 00:03:00 .
3m
Withdraw supernatant and resuspend in 100 µL of lysis buffer and incubate On ice for 00:05:00 .
5m
Sonicate the cells in the Bioruptor for 3 cycles of 00:00:30 on and 00:00:30 off at high power.
1m
Centrifuge the lysate at 500 x g for 00:05:00 and place the clarified lysate in a new 1.5 ml tube.
Note
This step is crucial to ensure the removal of high-diameter material (e.g. unlysed cells) that may clog the filter paper and non-specifically trap proteins on it.
Note
If desired, this is a convenient stopping point, wherein you can snap-freeze your lysate in liquid nitrogen and proceed with the rest of the assay at a later time.
5m
Determine the concentration of protein in each sample by Pierce Rapid Gold BCA Protein Assay.
Filter Trap
Filter Trap
10m
Soak the cellulose acetate membrane in wash buffer for > 00:10:00 .
10m
For each well that you run, prepare 1020 µL of the sample containing 10.2 - 51 ug of lysate, with the remaining volume consisting of lysis buffer.
Assemble the slot blot vacuum manifold apparatus with the cellulose acetate membrane.
Wash each well with 300 µL wash buffer, inspecting each well to ensure that the buffer can drain through it.
Note
Avoid using any non-functioning wells.
Load 1000 µL (10 - 50 ug) of sample to each well.
Once the sample has drained through the wells, wash each well 3 times with 300 µL of wash buffer.
When all of the washes have drained through the wells, trim the membrane and proceed with standard immunodetection methods.