Aug 05, 2024
Denaturation analysis of α-synuclein fibrils
- 1Duke Univeristy
- West lab protocols
Protocol Citation: Arpine Sokratian 2024. Denaturation analysis of α-synuclein fibrils. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3bmdg8j/v1
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: Working
We use this protocol and it's working
Created: January 23, 2024
Last Modified: August 05, 2024
Protocol Integer ID: 93932
Keywords: ASAPCRN
Funders Acknowledgements:
ASAP
Grant ID: 020527
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.
Protocol materials
Guanidine ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272
Sodium Dodecyl Sulfate (SDS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #436143
N-LauroylsarcosineMerck MilliporeSigma (Sigma-Aldrich)Catalog #61739
Thermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)Catalog #PI90411
Alexa-647-MJFR14-6-4-2AbcamCatalog ##ab216309
Safety warnings
Hazard Identification and Risk of Exposure to the
Hazards:
Inhalation or spread through food or drink that contain fibrils aerosols or fibrils.
Protective gloves, safety glasses and lab coat must always be used when handling anything that possibly could contain α-synuclein fibrils. Food or drink is strictly prohibited in any environment where α-syn fibrils are used.
Routes of Transmission: Prior to assigning containment requirements, it is imperative to
understand the routes of transmission.
Some issues to address:
- What are the exposure routes/risks of most concern:
Inhalation or spread through food or drink that contain fibril aerosols or fibirls accordingly. Fibrils possibly might reach the brain regions through the olfactory epithelium; Risk of accidental needlestick/droplet splash while handling fibrils for in vitro or in vivo work.
- What are the consequences of exposure (potential illness, etc)
Fibrils may be considered as infectious material. Minimum to no hazard is expected from α-syn protein. There is no evidence that transmission of fibrils can lead to development Parkinson’s disease. However, taking into account prion-like properties of α-syn fibrils should therefore be handled cautiously and wisely. Strictly recommended using disposable materials and Personal Protective Equipment (PPE) such as gloves, face mask, etc.
PRECAUTIONS:
Laboratory work where high concentration of fibrils (more than 300 uM) is needed must comply with biosafety level 2 (BSL2) containment as described in the current edition of the CDC/NIH’s
Biosafety in the Microbiological and Biomedical Laboratories: http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm
Sharps safety precautions:
The use of sharps (glass pipettes, glass slides and cover slips, scalpels and
lancets) should be eliminated, when possible. Appropriate precautions should be
taken to avoid percutaneous injuries. These items should be disposed of
immediately in a puncture-resistant sharps container. Bending, recapping or
clipping of needles is prohibited. As described in CDC’s sharps safety website:
https://www.cdc.gov/sharpssafety/index.html
Procedural Methods
and Materials:
- Laboratory work where high concentration of fibrils (more than 300 uM) is needed must comply with biosafety level 2 (BSL2) containment. This means all aerosol generating procedures must be performed within the biosafety cabinet.
- All the fibrils work involves using PPE, aerosol-tight centrifuges, water bath sonicator in a closed cabinet, homogenization of frozen brain samples using probe-tip sonicator under the hood (collection of protein fractions in BSL2 cabinets), chromatography equipment in a closed-door fridge, sealed plates, safe lock microfuge tubes (or tubes wrapped/sealed with parafilm), and use of filtered tips for pipettes. All personnel must strictly adhere to these procedures.
- Use of proper PPE as stated in the section below. Use of available N95 respirators is voluntary (same for the use of available sleeve protectors). Follow safety precautions for sharps (for e.g., to avoid accidental needle sticks) while working with PFFs in the lab and for doing in vivo work.
Personal Protective
Equipment (PPE): Appropriate PPE includes gloves, lab coat and safety glasses,
face mask (voluntary N95 respirator use and sleeve protectors), face / bench
top splash shield for specific procedures as stated above.
Methods to minimize personal exposure: Strictly adhere to sharps safety precautions using needles or any material that can potentially cause wounds. Use disposable supplies where possible. Use the minimal amounts of α-fibrils needed for an experiment. Keep fibrils in closed tubes. 10% of SDS solution in water must be used for decontaminating work areas. Do not use NaOH or Sodium Hypochlorite or ethanol. Do not leave samples containing fibrils unattended at the bench.
Methods to prevent the release of fibrils/protect workers from aerosols,
splashes, splatters: protective gloves and clothing always be always be worn when handling frozen vials. High concentration of fibrils(>1mg/mL) always be handled under Biosafety cabinet and containment caps will be used while centrifugation. Centrifuge cups will be opened inside a biosafety cabinet. Face shield or benchtop splash shield will be used when working at the open bench.
Specimen transport
and removal of material(s) from the laboratory: Transported in secondary
container (plastic/Styrofoam) in a closed box. The closed box is carried in a
bag.
Standard
microbiological methods: hand washing after removal of gloves and before
leaving the work area, no mouth pipetting, strictly no food or drink in
refrigerators where material is stored, no eating in work area.
Cleaning &
Disinfection: Work area must be
cleaned with 10% SDS in water. Wipes used must be immediately disposed into
biohazard waste container. Any piece of equipment or supplies that possibly
have been exposed to fibrils must be wiped with 10% of SDS.
Waste Generation and Disposal Methods: The solutions that contain α-syn fibrils must be
decontaminated with 10% of SDS in water for 30 minutes and be thrown as a
biohazard waste in a sealed container/bag (use a minimal volume of fibrils needed
for an experiment, do not generate large volumes of fibril-containing liquids).
Use small biohazard bags to collect tips and consumables of experiment
performed, appropriately tie neck of bag in single knot and place in into
secondary biohazard waste container.
Spill and Accident Response Procedure: Describe all emergency procedures including spill clean-up. Describedisinfectants and
environmental decontamination. (ex., Outside of a BSC: If spill is a
respiratory hazard, evacuate 30 minutes to allow aerosols to settle. Place absorbent towels over the spill, apply freshly
prepared 10% SDS solution to entire area of spill starting on the outer edges
and working inward, pick up sharp items with mechanical device (not hands),
place all clean-up materials in a biohazard bag)
Preparation of α-synuclein sonicated fibrils
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.
Protocol
NAME
Preparation of mouse and human α-synuclein fibrilsCREATED BY
Arpine Sokratian
Evaluated sonicated fibrils should be diluted in PBS to concentration of 5 mg/mL.
Preparation of denaturating agents
Preparation of denaturating agents
Prepare stock solutions of
6M Guanidine HCL Guanidine ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #G3272
10% SDS - Sodium Dodecyl Sulfate (SDS)Merck MilliporeSigma (Sigma-Aldrich)Catalog #436143
10% Sarkosyl - N-LauroylsarcosineMerck MilliporeSigma (Sigma-Aldrich)Catalog #61739
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
Reaction mix preparation
Thermo Scientific™ Low Protein Binding Collection Tubes (1.5 mL)VWR InternationalCatalog #PI90411
Calculate the reaction mix: 2 mg/mL concentration of α-synuclein in 50 µL 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
Protocol
NAME
Dynamic Light Scattering measurementsCREATED BY
andrew.west west
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 Alexa-647-MJFR14-6-4-2AbcamCatalog ##ab216309 at concentration 1:5000 to visualize aggregated forms of a-synuclein species after treatment with chemical compounds
Protocol
NAME
Slot-blot analysis of recombinant α-synuclein fibrilsCREATED BY
Arpine Sokratian