Sonication-induced fragmentation analysis of α-synuclein fibrils
Arpine Sokratian
Abstract
This protocol is designed to quantify the fragmentation pattern of full-length α-synuclein fibrils, induced by ultrasonic frequencies through sonication. To ensure consistency, we have adopted the indirect
water-bath sonication method, which offers a more controlled distribution of ultrasonic waves compared to the cup-horn technique. Our protocol involves multiple time-points of sonication, specifically at 1 minute, 2
minutes, 10 minutes, 30 minutes, and 1 hour of sonication. We recommend analyzing the sonication-induced fragmentation of α-synuclein fibrils using dynamic light scattering measurements and acquisition electron
micrographs for quantification of fibril length and size distribution
Steps
Amplification of α-synuclein fibrils
Prepare α-synuclein fibrils according to the protocol attached
Preparation of mouse and human α-synuclein fibrils
Concentration measurements
Measure concentration of fibrils according to the protocol step #5
Real time-quaking induced conversion assay (RT-QUIC)
Dilute fibrils to the concentration 5 mg/mL in PBS
Use protein-low binding tubes
Prepare 20 ul aliquots as estimated sonication time-points: three-five technical replicates for each experimental sample, three separate sonication runs.
Place first round of replicates into the tube holder for the water-bath sonicator
Equipment
| Value | Label |
|---|---|
| Cuphorn | NAME |
| Qsonica | BRAND |
| 431C2 | SKU |
| http://www.sonicator.com/ | LINK |
Set the amplitude to 30%, temperature to 10°C, adjust the water level in the water tank to align the liquid level in the tubes
Set the desired time for the first time-point (for example: 2 minutes)
Collect the tubes after sonication, transfer 2 uL from each sonicated/technical repeat sample into eppendorf tube and dilute 100 times, then measure size range of fibrils using DLS
Collect 30 acquisitions with the acquisition time set for 10 seconds.
See the protocol
Dynamic Light Scattering measurements
Continue collecting the samples from the remaining time-points then repeat with another set of replicates.
Analyze the size distribution of small fibrils (ranging from 10 to 100 nm) at multiple time points
