Fibrillation assay
This protocol describes how Amytracker can be utilized for fibrillation assays and detection of amyloids in liquid samples. As all Amytracker variants are highly fluorescent only when they are bound to their target, they are ideally suited for spectrophotometric analysis. We recommend to perform a titration to use Amytracker in the lowest concentration possible for your specific application. The experimental conditions used to induce protein misfolding and aggregation can vary considerably depending on the amyloidogenic protein or peptide. It is important to note that Amytracker fluorescence can vary depending on pH and ionic strength of the buffer. In this protocol, fibrillation of bovine insulin is performed in 2 M acetic acid and 0.5 M NaCl.
Materials and Reagents:
- Amytracker - DMSO
- Fibrillation solution: 2 M acetic acid and 0.5 M NaCl in deionized water
- Bovine insulin
- Deionized water
- 96-well microtiter plate with lid.
- Plate reader
Assay Procedure:
- Prepare a dilution series of Amytracker (1:500, 1:1000, 1:2500, 1:5000) in Fibrillation solution and include a blank control (Fibrillation solution without Amytracker).
- Prepare 10 mg/ml bovine insulin in Fibrillation solution.
- Fill 1x5wells with 50 µl Fibrillation solution.
- Fill 6x5wells with 50 µl Bovin Insuline solution.
- Fill the rest of the wells with deionized water to avoid evaporation.
- Add 50 µl of each Amytracker dilution incl. Blank to Row 1 & 2.
- Place the lid on the microtiter plate and read emission every 10 min for 5 hours in total.
- Every hour, remove the plate from the Reader and add Amytracker dilutions incl. Blank to the next row filled with Bovine Insulin solution.
Readout
- Make sure to read from the bottom of the plate. Note that excitation and emission are different for each Amytracker variant and excitation- and emission maximum can slightly vary for each target. For reference excitation and emission settings, see table below.