Feb 01, 2024

Public workspaceTau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader

DOI
dx.doi.org/10.17504/protocols.io.dm6gp3nw8vzp/v1
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Patricia Yuste-Checa
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gp3nw8vzp/v1
Protocol Citation: Patricia Yuste-Checa, F Ulrich Hartl 2024. Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp3nw8vzp/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: January 25, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 94537
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently monitor Tau aggregation by thioflavin T fluorescence in a plate reader.
Attachments
Tau agg monitored by ThT in a plate reader.docx
Tau agg monitored by...
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Materials
Buffers:

  • Heparin: Amount50 undetermined in water, Heparin sodium salt from porcine intestinal mucosa (Merck, H3393). Can be stored at Temperature4 °C . Prepare a fresh working dilution at Amount4.5 undetermined in water (Concentration250 micromolar (µM) ).
  • Concentration0.2 Molarity (M) MgCl2
  • Concentration1 millimolar (mM) Thioflavin T (ThT). Can be stored at Temperature-20 °C .

ReagentHeparin sodium salt from porcine intestinal mucosaMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3393
Reagent96 well half-area plate of black polystyrene with a clear bottomCorningCatalog #3881




Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
Mix reagents to a final concentration of Concentration10 micromolar (µM) Tau (TauRD), Concentration2.5 micromolar (µM) Heparin, Concentration2 millimolar (mM) MgCl2, Concentration10 micromolar (µM) ThT in 1x PBS Ph7.2 . Prepare a mix for 4.5 reactions per condition (per condition, four technical replicates in each plate). Final volume is Amount80 µL per well.
Note
Molecular or chemical chaperones can be included in the reaction in order to study their effect on Tau aggregation.

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 instead filled with water.
Seal the plate with parafilm to avoid evaporation.
Set the following parameters in a SPARK multimode microplate reader (TECAN) and start the reaction:
  • Fluorescence measurement: ThT signal, excitation 440 nm, emission 480 nm, (use gain regulation) measured every 2 minutes.
  • Temperature Temperature37 °C
  • Constant shaking (50 seconds linear shaking: amplitude 4.5 mm, frequency 420 rpm - 50 seconds orbital shaking: amplitude 1.5 mm, frequency 360 rpm).
Note
  • SPARK multimode microplate reader (TECAN) is highly recommended due to its high sensitivity and the gain regulation mode that increases the fluorescence detection window. When using other plate readers, the sample ThT signal easily gets saturated even when reducing initial gain to the minimum gain capacity.
  • Cysteine-free TauRD (Tau residues 244-371, C291A/P301L/C322A/V337M), rapidly aggregates under these conditions, reaching the ThT plateau after 2 h aggregation.