Thioflavin T Assay
Michael X. Henderon
Published: 2024-01-26 DOI: 10.17504/protocols.io.x54v9p4kqg3e/v1
Abstract
This protocol details Thioflavin T assay.
Attachments
Steps
Thioflavin T Assay
1. Ideally, the same dilution of monomer, PBS alone and a previous batch of PFFs should be runin parallel. Mouse PFFs have low ThT fluorescence. In this case 5µL PFFs can be diluted 1:10.
Resuspend fibril reaction. Fibrils will settle over time. Dilute 1.5µL of 5mg/mL fibrillization reaction 1:50 with PBS (total 75µL).
Note
2.
Assay each fibrillization in triplicate on the 96 well black assay plate. Dispense 20µL of diluted α- synuclein fibrils per well.
3.
Dilute 1millimolar (mM) Thioflavin T stock into PBS 1:1000 to obtain the required volume of Thioflavine T solution at a concentration of 1micromolar (µM).
4.
Dispense 180µL of 1micromolar (µM) Thioflvain T per well.
5.
Maintain plate at Room temperature for 1h 0m 0s in the dark.
6.
Read plate on a Spectrophotometer excitation 450 nm, emission 510 nm, cutoff 475 nm.
