Sponsored Content by AbcamDec 15 2017
Active alpha-synuclein protein monomers and aggregates allow Lewy body pathology to be easily induced in neurons. They are suitable for both in vivo and in vitro experiments.
Lewy bodies – the intraneuronal aggregates of alpha-synuclein protein – are characteristics of Lewy body dementia, Parkinson’s disease and other medical disorders. They are found in the regions of the brain responsible for motor control and result in a progressive decline in mental ability.
Figure 1. Alpha-synuclein fibril aggregate formation from the monomeric form.
The Advantages of Alpha-synuclein Proteins
- Distinguish active endogenous alpha-synuclein levels following the addition of aggregate or monomer proteins using thioflavin T (ThT) or a corresponding beta-sheet binding dye
- Induce Lewy body pathology in the experiment 1
- Explore both in vitro and in vivo applications (for example screen drug targets)
- Compare the results with alpha-synuclein aggregates or monomer controls
Assay Alpha-synuclein Proteins using Thioflavin T (ThT)
It is the active alpha-synuclein protein aggregate that seeds the development of novel alpha-synuclein aggregates from a pool of active alpha-synuclein protein monomers. The fluorescent dye ThT binds to beta sheet-rich structures, like those found in alpha-synuclein aggregates. Upon binding, the dye’s emission spectrum undergoes a red-shift and elevated fluorescence intensity.
Products
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Product name
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Ab ID
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Recombinant human alpha-synuclein protein monomer (active)
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ab218818
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Recombinant human alpha-synuclein protein aggregate (active)
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ab218819
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Recombinant human alpha-synuclein protein monomer (control)
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ab218816
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Recombinant human alpha-synuclein protein aggregate (control)
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ab218817
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Thioflavin T (ThT)
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ab120751
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Alpha-synuclein Protein Assay Protocol
Procedure for assaying the binding of active alpha-synuclein aggregate and monomer to thioflavin T:
- 1 mM stock solution of ThT is prepared in dH2O (this should be prepared fresh and filtered through a 0.2 μm syringe filter).
- The ThT in PBS (pH 7.4) is diluted so that the final concentration of ThT in each well is 25 μM.
- The alpha-synuclein (aggregate and monomer) aliquots is thawed on ice just prior to use.
- Either 100 μM monomer or 10 nM aggregate (or both) is added to the appropriate wells.
- The plate is sealed and placed in a shaking incubator (600 rpm) at 37 °C.
- Using a fluorescence microplate reader (excitation 450 nm, emission 485 nm), the ThT fluorescence is measured from 1 hour up to 72 hours at 37 °C.
Emission curves of ThT show increased fluorescence (associated with the aggregation of alpha-synuclein protein) over time, when 100 µM of active alpha-synuclein monomer (ab218818) (light blue) is integrated with 10 nM of active alpha-synuclein aggregate (ab218819), as opposed to when 10 nM of control alpha-synuclein aggregate (purple line) is integrated with 100 µM of active alpha-synuclein monomer, or 10 nM of control alpha-synuclein aggregate (ab218817) (dark blue) is integrated with 100 µM of control alpha-synuclein monomer (ab218816). ThT ex = 450 nm, em = 485 nm.
Further Reading
- Luk, K. C. & Lee, V. M.-Y. Modeling Lewy pathology propagation in Parkinson’s disease. Parkinsonism Relat. Disord. 20, S85–S87 (2014).
- Vieira, B. D. M., Radford, R. A., Chung, R. S., Guillemin, G. J. & Pountney, D. L. Neuroinflammation in Multiple System Atrophy: Response to and Cause of α-Synuclein Aggregation. Front. Cell. Neurosci. 9, 437 (2015).
- Roberts, H. & Brown, D. Seeking a Mechanism for the Toxicity of Oligomeric α-Synuclein. Biomolecules 5, 282–305 (2015).
- Fernagut, P.-O. & Chesselet, M.-F. Alpha-synuclein and transgenic mouse models. Neurobiol. Dis. 17, 123–130 (2004).
- Ariesandi, W., Chang, C.-F., Chen, T.-E. & Chen, Y.-R. Temperature-Dependent Structural Changes of Parkinson’s Alpha-Synuclein Reveal the Role of Pre-Existing Oligomers in Alpha-Synuclein Fibrillization. PLoS One 8, e53487 (2013).
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