Mar 27, 2025

Public workspaceAmyloid beta (M1-42) Aggregation Monitored by Thioflavin-T (ThT) Fluorescence in a Plate Reader

DOI
dx.doi.org/10.17504/protocols.io.bp2l68ky5gqe/v1
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Patricia Yuste-Checa
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DOI: dx.doi.org/10.17504/protocols.io.bp2l68ky5gqe/v1
Protocol Citation: Patricia Yuste-Checa, F Ulrich Hartl 2025. Amyloid beta (M1-42) Aggregation Monitored by Thioflavin-T (ThT) Fluorescence in a Plate Reader. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l68ky5gqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 20, 2025
Last Modified: March 27, 2025
Protocol Integer ID: 124796
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol is based on Linse S. Methods Mol Biol. (2020), section “3.10 Preparation of Samples for Kinetics” and details how to efficiently monitor Aβ(M1–42) aggregation by thioflavin T fluorescence in a plate reader.
Materials
Buffers:
  • Gu6: pH Ph8.5
AB
Guanidinium hydrochloride6 M
Sodium phosphate20 mM
  • PE 8.5: pH Ph8.5
AB
Sodium phosphate20 mM
EDTA0.2 mM
  • Concentration1 millimolar (mM) Thioflavin T (ThT) in water. This can be stored at Temperature-20 °C .


Equipment

  • CLARIOstar plate reader (BMG Labtech)

ReagentCorning® 96-well Half Area Black/Clear Flat Bottom Polystyrene NBS Microplate, 25 per Bag, without LCorningCatalog #3881

Size Exclusion Chromatography
Size Exclusion Chromatography

Note
To discard any possible aggregate that could have formed during the freezing lyophilization process, size exclusion chromatography of the purified peptide should be performed before the kinetic experiment.
Dissolve Amount0.5 mg of lyophilized Aβ (M1–42) in Amount700 µL Gu6 buffer.
Note
See protocol “Purification of recombinant Amyloid beta peptide (M1-42) from Escherichia coli”.


Load the sample onto a Cytiva Superdex 75 Increase 10/300 GL (Amount24 mL ) SEC column previously equilibrated with PE 8.5 buffer.

Run the column with PE 8.5 at Amount0.5 µL .
Note
NOTE: Collect the fractions preferably in low binding tubes.


Size exclusion chromatogram

Aβ(M1–42) monomers elute at around Amount14-15 mL (fractions around 27-31).

Measure absorbance of fractions at 205 nm wavelength using a UV spectrophotometer using PE 8.5 buffer as blank.
Note
NOTE: Aβ (M1-42) contains just one tyrosine and no tryptophan. Therefore, measurement at 280 nm wavelength of low concentrated peptide may not be accurate.

Pool fractions containing Aβ(M1–42) monomers and measure the concentration at A205nm.
Prepare aggregation reactions:
  • Mix reagents to a final concentration of Concentration4 micromolar (µM) Aβ(M1–42), Concentration10 micromolar (µM) ThT in PE 8.5 buffer.
  • Prepare a mix for 4.5 reactions per condition (quadruplicates in each plate as technical replicates), final volume Amount80 µL per well.
Note
Mix of Amount360 µL = Amount80 µL reaction x 4.5.
  • Concentration of Aβ(M1–42) and ThT should be tested empirically in order to achieve a final concentration in a range that provides a linear response of the fluorescence intensity versus fibril mass concentration, see Linse S. Methods Mol Biol. (2020).
  • Molecular or chemical chaperones can be included in the reaction in order to study their effect on Aβ(M1–42) aggregation.
  • Keep Aβ(M1–42) peptide as well as the 96 well plate during preparation always on ice.


Mix
Dispense Amount80 µL of the mix into a well of a 96-well half-area plate of black polystyrene with a clear bottom (Corning 3881).

If possible, the outer wells should not be used and should be filled with water.
Seal the plate with parafilm to avoid excessive evaporation.
In a CLARIOstar plate reader (BMG Labtech) set the following parameters and start the reaction:

  • Fluorescence measurement: ThT signal, excitation 440 nm, emission 480 nm, measured every Duration00:03:00 .
  • Temperature Temperature37 °C .
Note
NOTE: Under these conditions Aβ(M1–42) rapidly aggregates, reaching a fluorescence plateau after approximately Duration02:00:00 aggregation. The data can be fitted using Sigma plot software (Sigmoidal, Sigmoid, 3 Parameter function) to obtain the half time for reaching the aggregation plateau.




Protocol references
Linse S. Expression and Purification of Intrinsically Disordered Aβ Peptide and Setup of Reproducible Aggregation Kinetics Experiment. Methods Mol Biol. 2020;2141:731 754. doi: 10.1007/978-1-0716-0524-0_38. PMID: 32696387.