Jan 25, 2024

Public workspaceThioflavin T Assay

DOI
dx.doi.org/10.17504/protocols.io.x54v9p4kqg3e/v1
  • Michael X. Henderon1
  • 1Van Andel Institute
Michael Henderson
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9p4kqg3e/v1
Protocol Citation: Michael X. Henderon 2024. Thioflavin T Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9p4kqg3e/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: December 20, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 93622
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
This protocol details Thioflavin T assay.
Attachments
932-2408.docx
932-2408.docx
17KB
Guidelines
Adapted from Alex Crowe, Jing Guo, Dustin Covell 032012 protocol, Mian Horvath Updates
Materials

Consumables:

  • 1 mM Thioflavin T in MilliQ
  • PBS
  • Equipment needed: Spectrophotometer
  • Black 96 well plate
Thioflavin T Assay
Thioflavin T Assay
1h
Resuspend fibril reaction. Fibrils will settle over time. Dilute Amount1.5 µL of Amount5 mg/mL fibrillization reaction 1:50 with PBS (total Amount75 µL ).

Note
Ideally, the same dilution of monomer, PBS alone and a previous batch of PFFs should be run
in parallel. Mouse PFFs have low ThT fluorescence. In this case Amount5 µL PFFs can be diluted 1:10.


Pipetting
Assay each fibrillization in triplicate on the 96 well black assay plate. Dispense Amount20 µL of diluted α- synuclein fibrils per well.

Pipetting
Dilute Concentration1 millimolar (mM) Thioflavin T stock into PBS 1:1000 to obtain the required volume of Thioflavine T solution at a concentration of Concentration1 micromolar (µM) .

Pipetting
Dispense Amount180 µL of Concentration1 micromolar (µM) Thioflvain T per well.

Pipetting
Maintain plate at TemperatureRoom temperature for Duration01:00:00 in the dark.

1h
Read plate on a Spectrophotometer excitation 450 nm, emission 510 nm, cutoff 475 nm.