Mar 18, 2025
Protocol for α-Synuclein Purification and Ionic Strength Modification Pivotal to High Yield and Reproducibility
- Chelva Janarthanam1,
- Griffin Clabaugh1,
- Manikandan Samidurai1,
- Zerui Wang2,
- Ileia Scheibe1,
- Huajun Jin1,
- Vellareddy Anantharam1,
- Ramona J B Urbauer3,
- Jeffrey L Urbauer4,
- Arthi Kanthasamy1,
- Xuemei Huang5,
- Vincenzo Donadio6,
- Wenquan Zou2,
- Anumantha G Kanthasamy1,
- Bradley Melvin7,
- Bradley R Melvin7
- 1Isakson Center for Neurological Disease Research, Department of Physiology and Pharmacology, University of Georgia, Athens, GA 30602, USA.;
- 2Department of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.;
- 3Department of Chemistry, University of Georgia, Athens, GA 30602, USA.;
- 4Department of Chemistry, University of Georgia, Athens, GA 30602, USA;
- 5Department of Neurology, Pennsylvania State University College of Medicine, Hershey, PA 17033, USA;
- 6IRCCS Institute of Neurological Sciences of Bologna, Complex Operational Unit Clinica Neurologica, 40138 Bologna, Italy.;
- 7Carver College of Medicine
Protocol Citation: Chelva Janarthanam, Griffin Clabaugh, Manikandan Samidurai, Zerui Wang, Ileia Scheibe, Huajun Jin, Vellareddy Anantharam, Ramona J B Urbauer, Jeffrey L Urbauer, Arthi Kanthasamy, Xuemei Huang, Vincenzo Donadio, Wenquan Zou, Anumantha G Kanthasamy, Bradley Melvin, Bradley R Melvin 2025. Protocol for α-Synuclein Purification and Ionic Strength Modification Pivotal to High Yield and Reproducibility. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbnd63gpk/v1
Manuscript citation:
Janarthanam, C.; Clabaugh, G.; Wang, Z.; Melvin, B.R.; Scheibe, I.; Jin, H.; Anantharam, V.; Urbauer, R.J.B.; Urbauer, J.L.; Ma, J.; et al. High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility. Int. J. Mol. Sci. 2024, 25, 5988. https://doi.org/10.3390/ijms25115988
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 18, 2025
Last Modified: March 18, 2025
Protocol Integer ID: 124533
Keywords: α-synuclein, Purification, SIZE-EXCLUSION, ION exchange
Abstract
Here we describe a protocol for purifying a high yield αSyn protein
purification protocol for the efficient production of monomers with a low
propensity for self-aggregation. Alpha-synuclein seed amplification assays
(αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson’s disease
(PD) by detecting misfolded αSyn and amplifying the signal through cyclic
shaking and resting in vitro. The ultra sensitivity of the assay affords the
ability to detect minute quantities of αSyn in peripheral tissues, but it also
presents various technical challenges in controlling batch-to-batch variability.
To address the problem of variability, we expressed wild type αSyn in BL21 Escherichia
coli, lysed the cells using osmotic shock, and isolated αSyn using acid
precipitation and fast protein liquid chromatography (FPLC). Following
purification, we optimized the ionic strength of the reaction buffer to better distinguish
the fluorescence maximum (Fmax) separation between disease and healthy control
tissues for enhanced assay performance. Our protein purification protocol
yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture).
Together, these methods are highly reproducible with high purity, stability and
yield.
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