Thioflavin T assay
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The document discusses the use of Thioflavin T (ThT) to detect amyloid fibrils. ThT is a fluorescent dye that exhibits enhanced fluorescence when bound to amyloid fibrils. This allows it to be used to diagnose amyloid fibrils in vitro and ex vivo. When bound to fibrils, ThT has an emission peak around 480nm with excitation at 440nm, whereas unbound ThT has different excitation and emission properties. The fluorescence of ThT can be used to monitor the formation of amyloid fibrils over time.
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- 1. Binding of Thioflavin to Amyloid Fibrils Mr.Halavath Ramesh Department of Chemistry Loyola College-Chennai University of Madras
- 2. Many proteins and peptides are able to self-assemble in solution in vitro and in vivo to form Amyloid like fibrils. Misfolding and aggregation of proteins into Amyloid fibrils. Protein fibrils are also called Amyloid aggregates, composed of insoluble proteins. They arise from at least 18 in appropriately folded version of proteins and polypeptides present naturally in the body. These misfolded structures alter their proper configuration such that they erroneously interact with one another or other cell components forming insoluble fibrils.
- 3. They have been associated with the pathology of more than 20 serious human diseases in that abnormal accumulation of Amyloid fibrils in organs may lead to amyloidosis, and may play a role in various neurodegenerative dis orders. They are self-associating highly repetitive polymers consisting of arrays of β-sheets that are held together via hydrogen bonding along the peptide backbone. Protein fibrils conformation tests: 1. The Congo Red assay 2. Thioflavin T Spectroscopic assay 3. ANS binding Assay 4. BSB[(trans, trans)-1-Bromo-2,5-bis-(3-hydroxy carbonyl -4-hydroxy)Styryl benzene]
- 4. 1.Congo Red Assay Congo red is an organic compound, the sodium salt of 3,3′-([1,1′-biphenyl]- 4,4′-diyl)bis(4-aminonaphthalene-1-sulfonic acid). It is an azo dye. Congo red is water-soluble, yielding a red colloidal solution; its solubility is greater in organic solvents. Staining with congo red (CR) is a qualitative method used for the identification of amyloids in vitro and in tissue sections.
- 5. Congo red is used for the visual detection of Amyloid in muscle and nerve fresh section in patients who have amyloidosis. The specificity of this staining results from Congo red's affinity for binding to fibril proteins enriched in β-sheet conformation.
- 6. 2.Thioflavin T Spectroscopic assay ThioflavinT (ThT) has been widely used to investigate Amyloid formation since 1989.ThT still continues to be a very valuble tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. ThT also known a basic yellow colour is a benzothiozole dye. Thioflavin T consists of a dimethylated benzothiozole ring connected to a dimethyl amino benzyl ring through at single C-C bond.
- 7. Thioflavin T is a benzothiozole dye that exhibits enhanced fluorescence upon binding to Amyloid fibrils and is commonly used to diagnose Amyloid fibrils both ex vivo and in vitro. Formation of Amyloid fibrils underlies a wide range of human disorders including Alzheimer’s and prion diseases. The amyloid fibrils can be readily detected thanks to Thioflavin T , a small molecule that gives strong fluorescence upon binding to amyloids.
- 20. ThT study is very confusing as I feel. There we need to see the emission of free and bound. They both have different excitation and emission. Bound one gives emission around 480nm if you excite at 440nm. The shift occurs due to binding. But here another issue will be the slit width. If it is lower , you will find a structural emission but in higher slit(5/10) you will find a clear single emission peak. ThT fluorescence to monitor fibrillation. Amyloid fibers are likely bound to the surface of the cuvette from a previous experiments. SDS is often not sufficient to remove them. Try washing in aqua regia. Many of factors can be the real culprit. The starting concentration of ThT in your reaction should be 20-25 uM.
- 21. Intense effort to detect , diagnose, and analyze the kinetic and structural properties of amyloid fibrils have generated a powerful toolkit of Amyloid specific molecular probes. Since its first description in 1959 , the fluorescent dye Thioflavin T (ThT) has become among the most widely used” gold standards “ for selectively staining and identifying Amyloid fibrils both in vivo and in vitro. The large enhancement of its fluorescence emission upon binding to fibrils makes ThT a particularly powerful and convenient tool.
- 22. ThT study is very confusing assay. There we need to see the emission of free and bound. They both have different excitation and emission. Bound one give emission around 480nm if you excite at 440nm. The shift occurs due to binding. But here another issue will be the slit width. If it is lower, you will find a structural emission but in higher slit(5/10) you will find a clear single emission peak. ThT fluorescence to monitor fibrillation.