Mar 03, 2025
Extraction of alpha-synuclein filaments from brain
- 1Van Andel Research Institute
Protocol Citation: Yang Yang 2025. Extraction of alpha-synuclein filaments from brain. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9z25v5r/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: February 28, 2025
Last Modified: March 03, 2025
Protocol Integer ID: 123601
Disclaimer
No
Abstract
The accumulation of α-synuclein filaments is a hallmark of neurodegenerative disorders such as Parkinson’s disease and dementia with Lewy bodies. Structural and biochemical characterization of these filaments is crucial for understanding their role in disease progression. Here, we present a detailed protocol for the extraction of α-synuclein filaments from postmortem human and animal brain tissue, enabling downstream structural analysis using cryo-electron microscopy (cryo-EM) and biochemical assays. This method involves a series of steps, including tissue homogenization, differential centrifugation, and detergent-based fractionation to selectively enrich for filamentous α-synuclein. The protocol ensures minimal disruption to filament morphology while effectively separating α-synuclein aggregates from other cellular components. This approach facilitates the study of strain-specific conformations, interactions with co-factors, and potential therapeutic targeting of pathogenic α-synuclein assemblies.
Extraction of alpha-synuclein filaments from brain
Extraction of alpha-synuclein filaments from brain
Homogenize and release the filaments from the tissue.
Cut and weight the tissue (0.2g-0.5g).
Tissues were homogenized in 20 vol(v/w) extraction buffer of 10 mM Tris-HCl, pH 7.5, 0.8 M NaCl, 10-20% sucrose and 1 mM EGTA.
Homogenates were brought to 2% sarkosyl and incubated for 60 min at 37 °C.
Following 10 min centrifugation at 10,000g, the supernatants were spun at 100,000g (45,000rpm) for 40-60 min.
Further enrich for filamentous α-synuclein and remove other components.
The pellets were resuspended in 500 μl/g extraction buffer and centrifuged at 3,000g for 5 min.
The supernatants were diluted threefold in buffer 2: 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl, 10% sucrose and 0.2% sarkosyl, and spun at 45,000 rpm for 30 min.
Resuspend the pellet with buffer 3 (100 ul/g tissue). Current concentration is for Cryo-EM, 1:10 dilution for Western Blot or negative staining.
For further analysis, the samples were centrifuged at 3,000 g for 2 min.
Protocol references
Yang Y, Shi Y, Schweighauser M, Zhang X, Kotecha A, Murzin AG et al. (2022) Structures of a-synuclein
filaments from human brains with Lewy pathology. Nature 610: 791-795.