Aug 22, 2022
Version 1
Preparation of α-synuclein fibrils amplified from clinical material V.1
This protocol is a draft, published without a DOI.
- 1Duke University
Protocol Citation: arpine.sokratian 2022. Preparation of α-synuclein fibrils amplified from clinical material. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 27, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63304
Keywords: ASAPCRN, synuclein fibrils from clinical tissue, synuclein fibril, synuclein, optional conjugation step with phrodo stp ester dye, phrodo stp ester dye, preparation of alpha, clinical material this protocol, dye
Abstract
This protocol describes the preparation of alpha-synuclein fibrils from clinical tissue with a quality control.
Optional conjugation step with phRodo STP ester dye is indicated
Materials
Eppendorf tubeProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
PBS PBS Thermo Fisher ScientificCatalog #28374
pHrodo™ iFL Red STP Ester (amine-reactive)Thermo FisherCatalog #P36011
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301 Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Protocol materials
Protein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
PBS Thermo Fisher ScientificCatalog #28374
pHrodo™ iFL Red STP Ester (amine-reactive)Thermo FisherCatalog #P36011
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Thermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)Catalog #PI90411
Thaw down an aliquot of alpha-synuclein monomer stock (track down a batch number with identified EU number, concentration, A260/280 ratio) on ice
Spin down an aliquot of alpha-synuclein monomer stock solution (20000 x g , 00:10:00 4 °C ) and measure the concentration using nanodrop
Add 3 µL of 10x diluted aliquot in PBS onto nanodrop pedestal;
Parameters: other proteins; coefficient extinction: 5.98; MW: 14.4 kDA
Perform two measurements and confirm <10% standard error between two measurements
If necessary, prepare 20X and 30X dilutions to confirm findings.
Equipment
NanoDrop™ One/OneC Microvolume UV-Vis Spectrophotometer
NAME
UV-Vis Spectrophotometer
TYPE
Thermo Scientific
BRAND
ND-ONE-W
SKU
10m
Calculate the reaction mix: 5 mg/mL concentration of alpha-synuclein in 200 µL in PBS (use safe-lock eppendorf tubesProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116 or other Thermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)Catalog #PI90411 )
Prepare a reaction mix containing 10% of clinical tissue and 5 mg/mL of alpha-synuclein in PBS
Incubate the reaction at 1000 rpm, 37°C for 120:00:00 using program settings: 1 min ON; 1 min OFF
Equipment
Eppendorf Thermomixer C Model 5382
NAME
Thermomixer C
TYPE
Eppendorf
BRAND
5382000023
SKU
Equipment
ThermoTop®
NAME
Smart block
TYPE
Eppendorf
BRAND
5308000003
SKU
5d
Spin down the insoluble fraction at 15000 rpm, 10°C, 00:10:00 ; take 100 µL of supernatant and add 1 mL of fresh PBS to the pellet
Note
Example of reaction mix after 5 days of incubation
10m
Gently resuspend and spin down the fibrils at 15000 rpm, 10°C, 00:10:00 ; take 100 µL of supernatant and add 1 mL of fresh PBS to the pellet (repeat 3 times)
10m
Conjugation of full-length fibrils with pHrodo dye3 steps
1. Dissolve 100 ug of dye in 50 ul of sterile DMSO (2 mg/mL)
2. Dilute fibrils to 1mg/mL concentration in PBS containing 0.1M bicarbonate (total volume: 0.95 mL)
3. Add 50 ug of dissolved dye to each reaction tube
4. Incubate overnight in eppendorf tube (foil-wrapped) with continuous shaking
5. In the morning, spin down the dye-fibril solution at 10,000 g for 10 min at 10C
6. Transfer the supernatant and dissolve the pellet with 1 mL of PBS (repeat 5 times)
Take out 900 ul of PBS leaving 100 ul of fibril pellet in the tubes. Transfer the fibrils into 0.6 mL PCR tubes (thick wall).
Sonicate the fibrils using water bath sonicator at 10C, 30% amplitude and for 1 hour (no OFF ON cycles). Check the level of water in the water-bath is in a line with the tube content.
Measure size of sonicated particles using DLS
