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Thioflavin T Assay for Alpha-synuclein Detection
Licia Miller Product Manager
α-Synuclein is a small molecule protein mainly found in the presynaptic terminals of the central nervous system. It consists of 140 amino acids and has a molecular weight of about 19 kDa. Under physiological conditions, it mainly exists in the form of soluble monomers, but under pathological conditions, α-synuclein will misfold and aggregate to form oligomers and fibers with β-folded structures, eventually leading to the formation of Lewy bodies, which are closely related to neurodegenerative diseases such as Parkinson's disease.
The aggregation process of α-synuclein may spread between cells, induce protein aggregation in neighboring cells, and aggravate neuronal damage. Studying its aggregation mechanism and detection method is of great significance for understanding the occurrence and development of diseases and developing diagnostic methods.
Currently, there are various methods for detecting α-synuclein, including enzyme-linked immunosorbent assay (ELISA), seed amplification assay (SAA), fluorescent dye method (such as thioflavin T), single-molecule fluorescence imaging technology, and circular dichroism (CD). These methods can be used to detect the level of α-synuclein in body fluids, monitor its aggregation process and study its dynamic behavior in cells, providing strong support for disease diagnosis and drug development.
This article mainly introduces the experimental protocol for the determination of α-synuclein using the thioflavin T (ThT) fluorescent dye method. ThT is a fluorescent dye that can specifically bind to α-synuclein polymers with a β-folded structure, and the fluorescence intensity is significantly enhanced after binding. It is often used to monitor the aggregation process of α-synuclein in vitro and is suitable for studying aggregation kinetics.
Experimental Steps
1. Prepare 1 mM Thioflavin T stock solution in dH2O, freshly prepared and filtered through a 0.2 μm syringe filter.
2. Dilute the Thioflavin T stock solution with PBS (pH 7.4) to ensure that the final concentration of Thioflavin T in each well is 25 μM (volume per well = 100 μL).
3. Thaw the alpha-synuclein sample to room temperature in advance.
4. Add 10 µM aggregates or 100 µM monomers (or a mixture of the two) to the corresponding wells. Use a pipette to pipette up and down to mix.
Note: The concentrations here are estimates and may require further optimization to obtain good signals for different experiments or samples.
5. Seal the plate and place it in a shaking incubator, 600 rpm, incubate at 37°C.
6. Use a fluorescence microplate reader to detect the fluorescence signal, set the excitation wavelength to 450 nm and the emission wavelength to 485 nm. Read the fluorescence intensity regularly during the incubation process.
Read at 37°C from 1 hour to 72 hours.
7. The increase in fluorescence intensity is proportional to the degree of aggregation of α-synuclein. By comparing the fluorescence intensity at different time points or under different treatment conditions, the aggregation dynamics of α-synuclein can be evaluated.
Note:
(1) Thioflavin T stock solution should be freshly prepared and filtered through a 0.2 μm syringe filter.
(2) α-synuclein samples should be thawed at room temperature before use and the concentration should be optimized to obtain a good signal.
(3) Particles or impurities that may exist in the sample may cause fluorescence signal scattering and reduce the signal-to-noise ratio. It is recommended to remove them by filtration or centrifugation.
(4) Fluorescent dyes have the problem of quenching. During the experiment, it is necessary to avoid light as much as possible to slow down fluorescence quenching.
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Categories: Protocols