Aug 21, 2022

Public workspacePreparation of α-synuclein fibrils amplified from clinical material V.1

This protocol is a draft, published without a DOI.
  • 1Duke University
Arpine Sokratian
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Protocol Citation: Arpine Sokratian 2022. Preparation of α-synuclein fibrils amplified from clinical material. protocols.io https://protocols.io/view/preparation-of-synuclein-fibrils-amplified-from-cl-b93gr8jw
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 26, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 63304
Keywords: ASAPCRN
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 tubeReagentProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
PBS ReagentPBS Thermo Fisher ScientificCatalog #28374
ReagentpHrodo™ iFL Red STP Ester (amine-reactive)Thermo FisherCatalog #P36011
ReagentDMSOSigma AldrichCatalog #472301 ReagentSodium bicarbonateSigma AldrichCatalog #S6014

Protocol materials
ReagentProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
Materials, Step 3
ReagentPBS Thermo Fisher ScientificCatalog #28374
Materials
ReagentpHrodo™ iFL Red STP Ester (amine-reactive)Thermo FisherCatalog #P36011
Materials
ReagentDMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Materials
ReagentSodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S6014
Materials
ReagentThermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)Catalog #PI90411
Step 3
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 (Shaker20000 x g , Duration00:10:00 Temperature4 °C ) and measure the concentration using nanodrop

Add Amount3 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: Concentration5 mg/mL concentration of alpha-synuclein in Amount200 µL in PBS (use safe-lock eppendorf tubesReagentProtein LoBind Tubes, 1.5 mLVWR InternationalCatalog #0030108116 or other ReagentThermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)VWR InternationalCatalog #PI90411 )

Prepare a reaction mix containing 10% of clinical tissue and Concentration5 mg/mL of alpha-synuclein in PBS

Incubate the reaction at Shaker1000 rpm, 37°C for Duration120:00:00 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
Incubation
Spin down the insoluble fraction at Centrifigation15000 rpm, 10°C, 00:10:00 ; take Amount100 µL of supernatant and add Amount1 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 Centrifigation15000 rpm, 10°C, 00:10:00 ; take Amount100 µL of supernatant and add Amount1 mL of fresh PBS to the pellet (repeat 3 times)
10m

Step case

Conjugation of full-length fibrils with pHrodo dye
3 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
Protocol
Dynamic Light Scattering measurements
NAME
Dynamic Light Scattering measurements
CREATED BY
andrew.west west