Mar 14, 2025

Public workspaceAlpha-synuclein Seed Amplification Assay

DOI
dx.doi.org/10.17504/protocols.io.kxygxwjj4v8j/v1
  • Sunil Srivastav1,
  • Davin Henderson2,
  • Jessika Suescun Ocampo1,
  • Divya Onkar Mondhe1,
  • * Richard Gordon1
  • 1School of Biomedical Science, Translational Research Institute, Queensland University of Technology, Brisbane, 4102, Australia;
  • 2CWD Evolution LLC, Fort Collins, CO, United States
Sunil Srivastav
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DOI: dx.doi.org/10.17504/protocols.io.kxygxwjj4v8j/v1
Protocol Citation: Sunil Srivastav, Davin Henderson, Jessika Suescun Ocampo, Divya Onkar Mondhe, * Richard Gordon 2025. Alpha-synuclein Seed Amplification Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwjj4v8j/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 have been using this protocol successfully, and it is working well.
Created: March 04, 2025
Last Modified: March 14, 2025
Protocol Integer ID: 123808
Keywords: seed amplification, alpha synculein
Abstract
Preparation of fecal samples for alpha-synuclein seed amplification assay.
Materials
1. RT-QuIC buffer
2. alpha-synuclein substrate
3. optic bottom 96 well plates
4. Fluorometer
Protocol
Protocol
Homogenize fecal samples in phosphate buffered saline
Centrifuge the homogenate and carefully remove the supernatant.
To the supernatant add metal-ion nanoparticles and resuspend them.
Next, perform magnetic separation of α-synuclein seeds.
Add these seeds to the 96 well plate and now resuspend them with Real-time quaking-induced conversion (RT-QuIC) buffer containing α-synuclein substrate.
Using a standard fluorometer, take fluorescence readings with an excitation of 450 nm and emission of 480 nm.
After a few hours are elapsed, an increase in fluorescence intensity could be observed, indicating alpha-synuclein seed amplification.
Protocol references
1. L. Concha-Marambio, S. Pritzkow, M.Shahnawaz, C. M. Farris, C. Soto, Seed amplification assay for the detection of pathologic alpha-synuclein aggregates in cerebrospinal fluid. Nat Protoc 18, 1179-1196 (2023).
2. D. M. Henderson et al., Detection of chronic wasting disease prion seeding activity in deer and elk feces by real-time quaking-induced conversion. J Gen Virol 98, 1953-1962 (2017).
3. N. D. Denkers, D. M. Henderson, C. K. Mathiason, E. A. Hoover, Enhanced prion detection in biological samples by magnetic particle extraction and real-time quaking-induced conversion. J Gen Virol 97, 2023-2029 (2016).