Denaturation analysis of α-synuclein fibrils
Arpine Sokratian
Abstract
This protocol details the specific parameters used to accurately measure the denaturation sensitivity of α-synuclein fibrils. Essential parts of this protocol describe the evaluation of sonicated fibrils to ensure the reproducibility of the results. Visualization of α-synuclein is recommended to be conducted using slot-blot analysis with aggregate-specific α-synuclein antibodies.
Steps
Preparation of α-synuclein sonicated fibrils
Follow the protocol to prepare the batch of sonicated fibrils. If the samples have been prepared earlier and kept at -80, thaw down in a water bath. Then, perform a quality control check to ensure the size distribution and concentration are corresponding to values outlined in the protocol attached.
Preparation of mouse and human α-synuclein fibrils
Evaluated sonicated fibrils should be diluted in PBS to concentration of 5 mg/mL.
Preparation of denaturating agents
Prepare stock solutions of
6M Guanidine HCL
10% SDS -
10% Sarkosyl -
Dilute the chemical solutions:
6M, 2M, 1M, 0.5M, 0.25M, 0.125M for GuHCL
2%, 0.2%, 0.02%, 0.002%, 0.0002%, 0.00002%
8%, 4%, 2%, 1%, 0.5%, 0.25%
Reaction mix preparation
Calculate the reaction mix: 2mg/mL concentration of α-synuclein in 50µL in reaction mix
Use protein low-binding tubes:
Reaction mix: 25 uL of chemical solution + 5 uL of PBS + 20 uL of 5 mg/mL sonicated fibrils
Once added, mix up very well
Prepare reaction mixes for each concentration of chemical solution in triplicates
Incubate at room temperature for 30 min
Evaluate the denaturation process using DLS analysis
Dynamic Light Scattering measurements
Dilute samples up to 200 ng/mL (10.000x) to reach 1 mL of the volume, vortex before each dilution cycle
Follow instruction for the slot-blot analysis,
use
Slot-blot analysis of recombinant α-synuclein fibrils
