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Alpha-synuclein protein assay

Protocol for assaying active alpha-synuclein monomer and aggregate binding to thioflavin T.
Last edited Wed 01 Feb 2023

ThT emission curves show increased fluorescence (correlated to alpha-synuclein protein aggregation) over time when 10 µM of active alpha-synuclein aggregate (ab218819) is combined with 100 µM of active alpha-synuclein monomer (ab218818) (light blue), as compared to when 100 µM of active alpha-synuclein monomer is combined with 10 µM of control alpha-synuclein aggregate (purple line), or 100 µM of control alpha-synuclein monomer (ab218816) is combined with 10 µM of control alpha-synuclein aggregate (ab218817) (dark blue). ThT ex = 450 nm, em = 485 nm.

Products used in this protocol

Product nameAbID
Recombinant human alpha-synuclein protein monomer (active) ab218818
Recombinant human alpha-synuclein protein aggregate (active)ab218819
Recombinant human alpha-synuclein protein monomer (control)ab218816
Recombinant human alpha-synuclein protein aggregate (control)ab218817
Thioflavin T (ThT)ab120751

Stage 1 - Method

Steps

1

Prepare 1 mM stock solution of Thioflavin T (ThT) in dH2O (prepare fresh and filter through a 0.2 μm syringe filter)

  • Prepare fresh in dH2O
  • Filter through a 0.2 μm syringe filter
2

Dilute the ThT in PBS (pH 7.4).

  • The final ThT concentration in each well is 25 μM
3

Thaw the alpha-synuclein aliquots on ice just before use.

  •  This includes both monomer and aggregate.
4

Add either 10 µM aggregate or 100 μM monomer to the appropriate wells.

5

Seal and place the plate in a shaking incubator at 37°C

  • Run the shaking incubator at 600rpm
6

Measure ThT fluorescence using a fluorescence microplate reader.

  • The reader should be set to excitation 450nm and emission 485nm.
  • Read at 37°C from 1 hour up to 72 hours.

Figure 1. Graph showing ThT fluorescence following interactions with alpha-synuclein monomers, aggregates and controls.