Alpha-synuclein is a neuronal protein implicated in synaptic function and neurodegenerative diseases such as Parkinson’s disease and Lewy body dementia. Alpha-synuclein assays are valuable for detecting changes in the early stages of neurodegenerative diseases, aiding in early diagnosis and intervention.

This alpha-synuclein assay protocol is designed to support researchers investigating the role of this protein in disease progression. These assays are used to study risk factors for Parkinson’s disease and other neurological disorders, including genetic mutations, environmental exposures, and mitochondrial function, which are relevant to Parkinson’s disease research.

Epidemiological research often uses lifetime risk to estimate the probability of developing neurodegenerative diseases. Research applications also include the study of spatial patterns and neuroimaging biomarker change, as well as additional biomarkers such as tau pathology, tau aggregation, and cerebrospinal fluid levels.

ThT is a fluorescent cell-permeable amyloid-binding benzothiazole salt. As a dye, it is used to visualise stacked β sheets in vitro  and  in vivo. The binding of ThT induces a shift in excitation maximum (385 nm to 450 nm) and emission maximum (445 nm to 482 nm). It is highly soluble in water and has limited ability to cross the blood-brain barrier.

ThT emission curves show increased fluorescence (correlated to alpha-synuclein protein aggregation) over time when 10 µM of active alpha-synuclein aggregate (ab218819) is combined with 100 µM of active alpha-synuclein monomer (ab218818) (light blue), as compared to when 100 µM of active alpha-synuclein monomer is combined with 10 µM of control alpha-synuclein aggregate (purple line), or 100 µM of control alpha-synuclein monomer is combined with 10 µM of control alpha-synuclein aggregate (ab218817) (dark blue).

Products used in this protocol

Method

Steps

Prepare 1 mM stock solution of Thioflavin T (ThT) in dH₂O (prepare fresh and filter through a 0.2 μm syringe filter).

• Prepare fresh in dH₂O
• Filter through a 0.2 μm syringe filter

Dilute the ThT in PBS (pH 7.4). The final ThT concentration in each well should be 25 μM (25 μL of the 1mM ThT solution to 975 μL of PBS).

Thaw the alpha-synuclein aliquots to room temperature just before use. This includes both monomer and aggregate.

Add either 10 µM aggregate or 100 μM monomer to the appropriate wells.

Seal and place the plate in a shaking incubator at 37°C. Run the shaking incubator at 600 rpm.

Measure ThT fluorescence using a fluorescence microplate reader.

• The reader should be set to excitation 450nm and emission 485nm.
• Read at 37°C from 1 hour up to 72 hours.

Role in neurodegenerative diseases

Neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, and other dementias, represent a growing global health challenge. These disorders are characterized by the progressive degeneration of neurons, leading to symptoms such as memory loss, cognitive decline, and motor dysfunction. The abnormal aggregation of proteins, such as tau and α-synuclein, is central to the pathogenesis of neurodegenerative diseases, which disrupts normal brain function. Understanding the mechanisms of protein aggregation is essential for developing effective disease-modifying therapies and reducing the global impact of dementia and related conditions. Ongoing disease studies continue to highlight the urgent need for new approaches to diagnosis, treatment, and prevention.

Protein aggregation and disease

Protein aggregation is a defining feature of many neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease. In these conditions, proteins like amyloid-β and α-synuclein misfold and accumulate, forming toxic protein aggregates that impair the function of brain cells. These aggregates can be detected in cerebrospinal fluid and, increasingly, in blood samples, making them valuable biomarkers for early diagnosis and disease monitoring. Research in mammalian cells has shown that protein aggregation not only contributes to neuronal dysfunction but also offers a target for new treatments aimed at halting or reversing disease progression. Sensitive assays, such as those detecting α-synuclein aggregates, are instrumental in advancing our understanding of protein aggregation and its role in neurodegenerative diseases, paving the way for improved diagnosis and therapeutic strategies.

Clinical relevance and diagnosis

Diagnosing neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease remains a complex process, often requiring a combination of clinical assessment, biomarker analysis, and advanced imaging. Biomarkers found in spinal fluid, such as amyloid-β and tau, along with blood samples, provide critical information about disease pathology and progression.

Comparison to other methods

While mass spectrometry offers detailed structural insights, it lacks the simplicity and accessibility of this protocol. This assay strikes a balance between precision and practicality, making it suitable for routine lab use and large-scale studies. Its compatibility with standard plate readers and minimal hands-on time further enhances usability.

Our alpha-synuclein ELISA kits offer a streamlined approach to quantifying alpha-synuclein in serum, plasma, and cell lysates, while the Thioflavin T assay provides insights into aggregation dynamics. These protocols are used in research involving Parkinson’s disease patients, in longitudinal study designs, and in the differential diagnosis of Parkinson’s disease and multiple system atrophy. They are also applied in studies of Alzheimer's disease and dementia and in genetic research on autosomal dominant Alzheimer's disease.

Applications

These alpha-synuclein assays are widely used in neurodegenerative disease research, particularly in studies of Parkinson’s disease, Alzheimer’s disease, and Lewy body dementia. The ELISA kit is ideal for quantifying alpha-synuclein in human serum, plasma, and cell extracts, supporting biomarker discovery and drug development. The Thioflavin T assay is suited for investigating protein aggregation kinetics, screening aggregation inhibitors, and validating therapeutic targets.

Limitations

While highly effective, these assays have some limitations. The Thioflavin T assay, though useful for aggregation studies, may not distinguish between different aggregate species or provide structural resolution. Additionally, fluorescence readings can be affected by plate reader settings and incubation conditions. Researchers should validate assay conditions for their specific sample types and experimental goals.

Troubleshooting

Inconsistent fluorescence may result from improper ThT preparation or temperature fluctuations. Always filter ThT solutions and maintain consistent incubation at 37°C. Aggregation variability can be minimized by using standardized recombinant proteins. If background noise is high, verify plate washing steps and reagent purity.

Abcam's ELISA kits should only be used for research purposes and should not be used for diagnostic purposes.