Jan 31, 2024

α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader

DOI
https://dx.doi.org/10.17504/protocols.io.8epv5x87dg1b/v1
  • Patricia Yuste-Checa1,
  • F Ulrich Hartl1
  • 1Department of Cellular Biochemistry, Max Planck Institute of Biochemistry
Patricia Yuste-Checa
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DOI: https://dx.doi.org/10.17504/protocols.io.8epv5x87dg1b/v1
Protocol Citation: Patricia Yuste-Checa, F Ulrich Hartl 2024. α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x87dg1b/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: 94477
Keywords: ASAPCRN, synuclein aggregation, synuclein, plate reader, thioflavin, fluorescence
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000282
Abstract
This protocol details how to efficiently monitor α-Synuclein aggregation by thioflavin T fluorescence in a plate reader.
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α-Synuclein agg moni...
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Materials
Buffers:

  • α-Synuclein fibril buffer:
AB
NaN30.05%
ThT10 μM
KCl150 mM
Tris-HCl pH 7.650 mM
  • 1 millimolar (mM) Thioflavin T (ThT). Can be stored at -20 °C .
  • 6.5% NaN3

96 well half-area plate of black polystyrene with a clear bottomCorningCatalog #3881


α-Synuclein aggregation monitored by thioflavin-T (ThT) fluorescence in a plate reader
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Mix reagents to a final concentration of 200 micromolar (µM) α-Synuclein, 0.05 % NaN3,
10 micromolar (µM) ThT in α-Synuclein fibril buffer. 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 α-Synuclein aggregation.

Dispense 80 µL of the mix into a well of a 96 well half-area plate of black polystyrene with a clear bottom.

If possible, the outer wells should not be used and should be filled them 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 00:10:00 .
  • Temperature 37 °C
  • Constant shaking (00:02:00 linear shaking: amplitude 1.5 mm, frequency 1080 rpm - 00:02:00 orbital shaking: amplitude 1 mm, frequency 510 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.
  • Under these conditions α-Synuclein (A53T) slowly aggregates, reaching the ThT plateau after approximately 50 h aggregation.







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