Jan 25, 2024
Thioflavin T Assay
- Michael X. Henderon1
- 1Van Andel Institute
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: September 20, 2024
Protocol Integer ID: 93622
Keywords: ASAPCRN, protocol
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020616
Abstract
This protocol details Thioflavin T assay.
Attachments
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
1h
Resuspend fibril reaction. Fibrils will settle over time. Dilute 1.5 µL of 5 mg/mL fibrillization reaction 1:50 with PBS (total 75 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 5 µL PFFs can be diluted 1:10.
Assay each fibrillization in triplicate on the 96 well black assay plate. Dispense 20 µL of diluted α- synuclein fibrils per well.
Dilute 1 millimolar (mM) Thioflavin T stock into PBS 1:1000 to obtain the required volume of Thioflavine T solution at a concentration of 1 micromolar (µM) .
Dispense 180 µL of 1 micromolar (µM) Thioflvain T per well.
Maintain plate at Room temperature for 01:00:00 in the dark.
1h
Read plate on a Spectrophotometer excitation 450 nm, emission 510 nm, cutoff 475 nm.