Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies
Abstract
:1. Introduction
2. Results
2.1. Differential Solubility of α-Synuclein in Selective Detergents
2.2. Sonication Increases α-Synuclein Solubility in SDS Buffer
2.3. Sonication Does Not Affect α-Synuclein Solubility in RIPA Buffer
2.4. α-Synuclein Is Fully Soluble in Polyethoxylate Detergents Irrespective of Sonication
3. Discussion
4. Materials and Methods
4.1. Fly Strains
4.2. Tissue Collection and Fractionation
4.2.1. Three-Step Fractionation Protocol
4.2.2. Two-Step Fractionation Protocol
4.3. Quantitative Western Immunoblotting
4.4. Experimental Design and Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Name | Composition | Manufacturer (Catalog Number) |
---|---|---|
TBS | 150 mM NaCl, 20 mM Tris base, pH = 7.6 | |
SDS | 5% w/v sodium dodecyl sulfate (SDS), 150 mM NaCl, 20 mM Tris base, pH = 7.6 | |
RIPA | 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 25 mM Tris HCl, pH = 7.6 | Thermo Fisher Scientific, Waltham, MA, USA (89900) |
NP-40 | 1% NP-40 (74385, Sigma-Aldrich), 150 mM NaCl, 20 mM Tris base, pH = 7.6 | |
Triton X-100 | 1% TritonX-100 (T9284, Sigma-Aldrich), 150 mM NaCl, 20 mM Tris base, 20 mM NaF, pH = 7.6 | |
urea/SDS | 8 M urea, 5% w/v SDS, 150 mM NaCl, 20 mM Tris base | |
4× Laemmli | 0.02% bromophenol blue, 4.4% lithium dodecyl sulphate, 44.4% glycerol, 277.8 mM Tris HCl, pH = 6.8 | BioRad, Hercules, CA, USA (1610747) |
TBST | 0.1% v/v Tween-20, 150 mM NaCl, 20 mM Tris base, pH = 7.4 |
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Imomnazarov, K.; Lopez-Scarim, J.; Bagheri, I.; Joers, V.; Tansey, M.G.; Martín-Peña, A. Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies. Int. J. Mol. Sci. 2024, 25, 3643. https://doi.org/10.3390/ijms25073643
Imomnazarov K, Lopez-Scarim J, Bagheri I, Joers V, Tansey MG, Martín-Peña A. Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies. International Journal of Molecular Sciences. 2024; 25(7):3643. https://doi.org/10.3390/ijms25073643
Chicago/Turabian StyleImomnazarov, Khondamir, Joshua Lopez-Scarim, Ila Bagheri, Valerie Joers, Malú Gámez Tansey, and Alfonso Martín-Peña. 2024. "Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies" International Journal of Molecular Sciences 25, no. 7: 3643. https://doi.org/10.3390/ijms25073643
APA StyleImomnazarov, K., Lopez-Scarim, J., Bagheri, I., Joers, V., Tansey, M. G., & Martín-Peña, A. (2024). Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies. International Journal of Molecular Sciences, 25(7), 3643. https://doi.org/10.3390/ijms25073643