Mar 30, 2023

Public workspaceSoluble and insoluble A-SYN fractionation

DOI
dx.doi.org/10.17504/protocols.io.rm7vzbrn2vx1/v1
  • 1German Center for Neurodegenerative Diseases (DZNE), Tübingen, 72076 Germany
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzbrn2vx1/v1
Protocol Citation: michela.deleidi, Hariam Raji, Federico Bertoli 2023. Soluble and insoluble A-SYN fractionation. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbrn2vx1/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: March 28, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 79526
Keywords: Soluble A-SYN fractionation, insoluble A-SYN fractionation, ASAPCRN
Abstract
Soluble/insoluble alpha-synuclein fractionation is a technique used to separate different forms of the alpha-synuclein protein based on their solubility properties.
Attachments
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Materials
Materials

Extraction buffer
AB
Triton X-1001%
NaCl150 mM
glycerol10%
HEPES pH 7.425 mM
EDTA1 mM
MgCl21.5 mM
  • 50 mM NaF
  • 2 mM NA3VO4
  • 0.5 mM PMSF
  • 50 mM Tris
  • cup horn probe sonicator (Qsonica – Q700)
Soluble and insoluble A-SYN fractionation
Soluble and insoluble A-SYN fractionation
1h 50m
1h 50m
Perform extraction and detection of Triton-soluble (T-sol) and Triton-insoluble (T-insol) alpha-synuclein as described in Stojkovska and Mazzulli(2021).
Lyse individual organoids in 1% Triton X-100 extraction buffer supplemented with 1X PIC, Concentration50 millimolar (mM) NaF, Concentration2 millimolar (mM) NA3VO4 and Concentration0.5 millimolar (mM) PMSF.

Extraction buffer
AB
Triton X-1001%
NaCl150 mM
glycerol10%
HEPES pH 7.425 mM
EDTA1 mM
MgCl21.5 mM
Digestion
Homogenize samples with a pestle and incubate on a platform shaker in an ice-water slurry for Duration00:30:00 , followed by three freeze/thaw cycles and ultracentrifugation at Centrifigation100000 x g, 4°C, 00:30:00 .

1h
Centrifigation
Collect the supernatant (Triton-X Soluble fraction).
Wash the remaining pellet in Triton X-100 extraction buffer followed by another ultracentrifugation at Centrifigation100000 x g .

Centrifigation
Wash
Resuspend the pellet in 2% SDS buffer containing Concentration50 millimolar (mM) Tris, Ph7.4 and 1X PIC, boil it for Duration00:10:00 at Temperature100 °C (Triton-X insoluble Fraction) and label the T-insol fraction.
10m
Sonicate Tx-Insoluble samples for Duration00:10:00 at 30% power, 20C in a cup horn sonicator (Qsonica-Q700), and then boil them again for Duration00:10:00 at Temperature100 °C .

20m
Ultracentrifuge Tx-Insoluble samples at Centrifigation100000 x g, 21°C, 00:30:00 .
30m
Centrifigation
Collect the supernatant (SDS-soluble fraction).
Detect protein concentrations using a BCA assay and load Amount30 µg of total protein for each condition.