Feb 01, 2024
Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
- Patricia Yuste-Checa1,
- F Ulrich Hartl1
- 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
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, tau aggregation, plate reader, fluorescence, thioflavin
Funders Acknowledgements:
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
Materials
Buffers:
- Heparin: 50 µL in water, Heparin sodium salt from porcine intestinal mucosa (Merck, H3393). Can be stored at 4 °C . Prepare a fresh working dilution at 4.5 µL in water (250 micromolar (µM) ).
- 0.2 Mass Percent MgCl2
- 1 millimolar (mM) Thioflavin T (ThT). Can be stored at -20 °C .
Heparin sodium salt from porcine intestinal mucosaMerck MilliporeSigma (Sigma-Aldrich)Catalog #H3393
96 well half-area plate of black polystyrene with a clear bottomCorningCatalog #3881
Tau aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
Mix reagents to a final concentration of 10 micromolar (µM) Tau (TauRD), 2.5 micromolar (µM) Heparin, 2 millimolar (mM) MgCl2, 10 micromolar (µM) ThT in 1x PBS 7.2 . Prepare a mix for 4.5 reactions per condition (per condition, four technical replicates in each plate). Final volume is 80 µL per well.
Note
Molecular or chemical chaperones can be included in the reaction in order to study their effect on Tau aggregation.
Dispense 80 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 37 °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.