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Thioflavin T
Thioflavin T (ThT), also called Basic Yellow 1 or CI 49005, is a benzothiazole salt. The dye is used to visualize plaques composed of beta-amyloid found in the brains of Alzheimer\'s disease patients. When it binds to beta sheets, such as those in amyloid oligomers, the dye undergoes a characteristic 120 nm red shift of its excitation spectrum that may be selectively excited at 450 nm, resulting in a fluorescence signal at 482 nm. ThT binds rapidly and specifically to the anti-parallel beta-sheet fibrils formed from synthetic beta-amyloid (1-40), but does not bind to monomer or oligomeric intermediates. The binding of ThT does not interfere with the aggregation of beta-amyloid peptide into amyloid fibrils. Thioflavin T, succinimidyl ester is a valuble tool for making various bioconjugates of Thioflavin T that can direct Thioflavin T to a desired target. It is an essential reagent to make the peptides and other conjugates of Thioflavin T.
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
1. Protein stock solution (Solution A)
Mix 100 µL of a reaction buffer (e.g., 1 M sodium carbonate solution or 1 M phosphate buffer with pH ~9.0) with 900 µL of the target protein solution (e.g. antibody, protein concentration >2 mg/mL if possible) to give 1 mL protein labeling stock solution. Note: The pH of the protein solution (Solution A) should be 8.5 ± 0.5. If the pH of the protein solution is lower than 8.0, adjust the pH to the range of 8.0-9.0 using 1 M sodium bicarbonate solution or 1 M pH 9.0 phosphate buffer. Note: The protein should be dissolved in 1X phosphate buffered saline (PBS), pH 7.2-7.4. If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS, pH 7.2-7.4, to remove free amines or ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation. Note: Impure antibodies or antibodies stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. The presence of sodium azide or thimerosal might also interfere with the conjugation reaction. Sodium azide or thimerosal can be removed by dialysis or spin column for optimal labeling results. Note: The conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL. For optimal labeling efficiency the final protein concentration range of 2-10 mg/mL is recommended.2. Thioflavin T, succinimidyl ester stock solution (Solution B)
Add anhydrous DMSO into the vial of Thioflavin T, succinimidyl ester to make a 10 mM stock solution. Mix well by pipetting or vortex. Note: Prepare the dye stock solution (Solution B) before starting the conjugation. Use promptly. Extended storage of the dye stock solution may reduce the dye activity. Solution B can be stored in freezer for two weeks when kept from light and moisture. Avoid freeze-thaw cycles.SAMPLE EXPERIMENTAL PROTOCOL
This labeling protocol was developed for the conjugate of Goat anti-mouse IgG with Thioflavin T, succinimidyl ester. You might need further optimization for your particular proteins. Note: Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity.
Run conjugation reaction
- Use 10:1 molar ratio of Solution B (dye)/Solution A (protein) as the starting point: Add 5 µL of the dye stock solution (Solution B, assuming the dye stock solution is 10 mM) into the vial of the protein solution (95 µL of Solution A) with effective shaking. The concentration of the protein is ~0.05 mM assuming the protein concentration is 10 mg/mL and the molecular weight of the protein is ~200KD. Note: We recommend to use 10:1 molar ratio of Solution B (dye)/Solution A (protein). If it is too less or too high, determine the optimal dye/protein ratio at 5:1, 15:1 and 20:1 respectively.
- Continue to rotate or shake the reaction mixture at room temperature for 30-60 minutes.
Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.- Prepare Sephadex G-25 column according to the manufacture instruction.
- Load the reaction mixture (From "Run conjugation reaction") to the top of the Sephadex G-25 column.
- Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
- Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate. Note: For immediate use, the dye-protein conjugate need be diluted with staining buffer, and aliquoted for multiple uses. Note: For longer term storage, dye-protein conjugate solution need be concentrated or freeze dried.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Thioflavin T, succinimidyl ester to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 176.239 µL | 881.197 µL | 1.762 mL | 8.812 mL | 17.624 mL |
| 5 mM | 35.248 µL | 176.239 µL | 352.479 µL | 1.762 mL | 3.525 mL |
| 10 mM | 17.624 µL | 88.12 µL | 176.239 µL | 881.197 µL | 1.762 mL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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Citations
View all 8 citations: Citation Explorer
Human Telomeric Hybrid-2-over-Hybrid-1 G-quadruplex Targeting and a Selective Hypersaline-Tolerant Sensor Using Abasic Site-Engineered Monomorphism
Authors: Wu, Tao and Ye, Meiyun and Mao, Tianyi and Lin, Fan and Hu, Yuehua and Gan, Ning and Shao, Yong
Journal: Analytica Chimica Acta (2017)
Authors: Wu, Tao and Ye, Meiyun and Mao, Tianyi and Lin, Fan and Hu, Yuehua and Gan, Ning and Shao, Yong
Journal: Analytica Chimica Acta (2017)
Fluorescent dyes in the context of DNA-binding: The case of Thioflavin T
Authors: Biancardi, Aless and ro , undefined and Biver, Tarita and Mennucci, Benedetta
Journal: International Journal of Quantum Chemistry (2017)
Authors: Biancardi, Aless and ro , undefined and Biver, Tarita and Mennucci, Benedetta
Journal: International Journal of Quantum Chemistry (2017)
Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels
Authors: Sugimoto, Shinya and Arita-Morioka, Ken-ichi and Mizunoe, Yoshimitsu and Yamanaka, Kunitoshi and Ogura, Teru
Journal: Nucleic acids research (2015): gkv338
Authors: Sugimoto, Shinya and Arita-Morioka, Ken-ichi and Mizunoe, Yoshimitsu and Yamanaka, Kunitoshi and Ogura, Teru
Journal: Nucleic acids research (2015): gkv338
Determinants of amyloid fibril degradation by the PDZ protease HTRA1
Authors: Poepsel, Simon and Sprengel, Andreas and Sacca, Barbara and Kaschani, Farnusch and Kaiser, Markus and Gatsogiannis, Christos and Raunser, Stefan and Clausen, Tim and Ehrmann, Michael
Journal: Nature chemical biology (2015): 862--869
Authors: Poepsel, Simon and Sprengel, Andreas and Sacca, Barbara and Kaschani, Farnusch and Kaiser, Markus and Gatsogiannis, Christos and Raunser, Stefan and Clausen, Tim and Ehrmann, Michael
Journal: Nature chemical biology (2015): 862--869
Mechanistic aspects of thioflavin-T self-aggregation and DNA binding: evidence for dimer attack on DNA grooves
Authors: Biancardi, A and Biver, T and Burgalassi, A and Mattonai, M and Secco, F and Venturini, M
Journal: Physical Chemistry Chemical Physics (2014): 20061--20072
Authors: Biancardi, A and Biver, T and Burgalassi, A and Mattonai, M and Secco, F and Venturini, M
Journal: Physical Chemistry Chemical Physics (2014): 20061--20072
References
View all 28 references: Citation Explorer
Methods for enhancing the accuracy and reproducibility of Congo red and thioflavin T assays
Authors: Eisert R, Felau L, Brown LR.
Journal: Anal Biochem (2006): 144
Authors: Eisert R, Felau L, Brown LR.
Journal: Anal Biochem (2006): 144
Characteristics of the binding of thioflavin S to tau paired helical filaments
Authors: Santa-Maria I, Perez M, Hern and ez F, Avila J, Moreno FJ.
Journal: J Alzheimers Dis (2006): 279
Authors: Santa-Maria I, Perez M, Hern and ez F, Avila J, Moreno FJ.
Journal: J Alzheimers Dis (2006): 279
Mechanism of thioflavin T accumulation inside cells overexpressing P-glycoprotein or multidrug resistance-associated protein: role of lipophilicity and positive charge
Authors: Darghal N, Garnier-Suillerot A, Salerno M.
Journal: Biochem Biophys Res Commun (2006): 623
Authors: Darghal N, Garnier-Suillerot A, Salerno M.
Journal: Biochem Biophys Res Commun (2006): 623
Oligo(ethylene glycol) derivatives of Thioflavin T as inhibitors of protein-amyloid interactions
Authors: Inbar P, Li CQ, Takayama SA, Bautista MR, Yang J.
Journal: Chembiochem (2006): 1563
Authors: Inbar P, Li CQ, Takayama SA, Bautista MR, Yang J.
Journal: Chembiochem (2006): 1563
Lipophilic analogs of thioflavin S as novel amyloid-imaging agents
Authors: Wu C, Cai L, Wei J, Pike VW, Wang Y.
Journal: Curr Alzheimer Res (2006): 259
Authors: Wu C, Cai L, Wei J, Pike VW, Wang Y.
Journal: Curr Alzheimer Res (2006): 259
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