Source 1primary paper1993Protein Science
Toolkit/Thioflavine T fluorometric assay for amyloid aggregation detection
Thioflavine T fluorometric assay for amyloid aggregation detection
Also known as: Thioflavine T (ThT) fluorometric technique, ThT fluorescence assay
Taxonomy: Technique Branch / Method. Workflows sit above the mechanism and technique branches rather than replacing them.
Summary
Thioflavine T (ThT) associates rapidly with aggregated fibrils ... giving rise to a new excitation maximum at 450 nm and enhanced emission at 482 nm ... This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.
Usefulness & Problems
Why this is useful
This assay uses Thioflavine T fluorescence to detect aggregated amyloid fibrils in solution by a binding-associated spectral shift and enhanced emission. The abstract presents it as a fluorometric technique for following amyloid aggregation.; detecting amyloid aggregation in solution; monitoring amyloid fibril assembly kinetics; testing agents that modulate amyloid assembly or disassembly
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This assay uses Thioflavine T fluorescence to detect aggregated amyloid fibrils in solution by a binding-associated spectral shift and enhanced emission. The abstract presents it as a fluorometric technique for following amyloid aggregation.
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detecting amyloid aggregation in solution
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monitoring amyloid fibril assembly kinetics
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testing agents that modulate amyloid assembly or disassembly
Problem solved
It provides a solution-phase readout for amyloid fibril formation and is proposed for kinetic studies of assembly and disassembly. It also offers a way to test candidate modulators of aggregation.; provides a fluorescence readout that distinguishes aggregated fibrils from non-aggregated peptide states
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It provides a solution-phase readout for amyloid fibril formation and is proposed for kinetic studies of assembly and disassembly. It also offers a way to test candidate modulators of aggregation.
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provides a fluorescence readout that distinguishes aggregated fibrils from non-aggregated peptide states
Problem links
provides a fluorescence readout that distinguishes aggregated fibrils from non-aggregated peptide states
LiteratureIt provides a solution-phase readout for amyloid fibril formation and is proposed for kinetic studies of assembly and disassembly. It also offers a way to test candidate modulators of aggregation.
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It provides a solution-phase readout for amyloid fibril formation and is proposed for kinetic studies of assembly and disassembly. It also offers a way to test candidate modulators of aggregation.
Taxonomy & Function
Primary hierarchy
Technique Branch
Method: A concrete measurement method used to characterize an engineered system.
Mechanisms
binding-associated spectral shiftdye binding to aggregated amyloid fibrilsfluorescence enhancementHeterodimerizationTechniques
Functional AssayTarget processes
No target processes tagged yet.
Implementation Constraints
cofactor dependency: cofactor requirement unknownencoding mode: genetically encodedimplementation constraint: context specific validationimplementation constraint: multi component delivery burdenoperating role: sensorswitch architecture: multi componentswitch architecture: recruitment
The method requires Thioflavine T, aggregated amyloid-forming peptide or protein samples, and fluorescence measurements of excitation and emission changes. The reported readout compares bound-dye and free-dye spectra.; requires Thioflavine T dye; requires aggregated fibrillar peptide or protein material to generate the reported signal; requires fluorescence measurement at the reported excitation and emission wavelengths
The abstract indicates it does not report on monomeric or dimeric peptide states because those do not react with ThT. It also does not preserve signal after guanidine-mediated aggregate dissociation.; does not react with monomeric or dimeric peptides; signal is destroyed by guanidine dissociation of aggregates; binding affinity differs between peptide substrates
Validation
Cell-freeBacteriaMammalianMouseHumanTherapeuticIndep. Replication
Supporting Sources
Ranked Claims
The Thioflavine T fluorometric technique should enable kinetic analysis of amyloid fibril assembly and testing of agents that modulate assembly or disassembly.
Thioflavine T fluorescence detects aggregated amyloid fibrils in solution through a binding-associated spectral shift and enhanced emission.
bound emission maximum 482 nmbound excitation maximum 450 nmfree dye emission maximum 445 nmfree dye excitation maximum 385 nm
Thioflavine T binds aggregated beta(1-28) with higher apparent affinity than aggregated beta(1-40).
Kd for beta(1-28) 0.54 microMKd for beta(1-40) 2 microM
High salt concentrations did not affect the reported Thioflavine T interaction, and a variety of polyhydroxy, polyanionic, or polycationic materials failed to interact or impede interaction with the amyloid peptides.
The Thioflavine T fluorescence change depends on the aggregated state because monomeric or dimeric peptides do not react and guanidine dissociation of aggregates destroys the signal.
Approval Evidence
1 source5 linked approval claimsfirst-pass slug thioflavine-t-fluorometric-assay-for-amyloid-aggregation-detection
Thioflavine T (ThT) associates rapidly with aggregated fibrils ... giving rise to a new excitation maximum at 450 nm and enhanced emission at 482 nm ... This fluorometric technique should allow the kinetic elucidation of the amyloid fibril assembly process as well as the testing of agents that might modulate their assembly or disassembly.
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application scopesupports
The Thioflavine T fluorometric technique should enable kinetic analysis of amyloid fibril assembly and testing of agents that modulate assembly or disassembly.
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assay capabilitysupports
Thioflavine T fluorescence detects aggregated amyloid fibrils in solution through a binding-associated spectral shift and enhanced emission.
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binding affinitysupports
Thioflavine T binds aggregated beta(1-28) with higher apparent affinity than aggregated beta(1-40).
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specificitysupports
High salt concentrations did not affect the reported Thioflavine T interaction, and a variety of polyhydroxy, polyanionic, or polycationic materials failed to interact or impede interaction with the amyloid peptides.
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state specificitysupports
The Thioflavine T fluorescence change depends on the aggregated state because monomeric or dimeric peptides do not react and guanidine dissociation of aggregates destroys the signal.
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Comparisons
Source-stated alternatives
The provided source does not name an alternative assay in the abstract. The web research summary notes related amyloid-binding dyes such as Congo red, X-34, and Pittsburgh compound B as nearby probe families.
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The provided source does not name an alternative assay in the abstract. The web research summary notes related amyloid-binding dyes such as Congo red, X-34, and Pittsburgh compound B as nearby probe families.
Source-backed strengths
rapid association with aggregated fibrils; distinct spectral shift upon binding aggregated fibrils; signal depends on aggregated state; high salt did not affect the interaction in the reported system
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rapid association with aggregated fibrils
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distinct spectral shift upon binding aggregated fibrils
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signal depends on aggregated state
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high salt did not affect the interaction in the reported system
Compared with higher-energy collisional dissociation
Thioflavine T fluorometric assay for amyloid aggregation detection and higher-energy collisional dissociation address a similar problem space.
Shared frame: same top-level item type; shared mechanisms: heterodimerization
Compared with reverse cross-saturation NMR methodology
Thioflavine T fluorometric assay for amyloid aggregation detection and reverse cross-saturation NMR methodology address a similar problem space.
Shared frame: same top-level item type; shared mechanisms: heterodimerization
Compared with top-down mass spectrometry
Thioflavine T fluorometric assay for amyloid aggregation detection and top-down mass spectrometry address a similar problem space.
Shared frame: same top-level item type; shared mechanisms: heterodimerization
Strengths here: looks easier to implement in practice.
Ranked Citations
- 1.