Jan 13, 2025

Public workspaceIn vitro coimmunoprecipitation between CHMP2B and alpha-synuclein

DOI
dx.doi.org/10.17504/protocols.io.8epv5rb66g1b/v1
  • 1Max Planck Institute of Biochemistry
Cole Sitron
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.8epv5rb66g1b/v1
Protocol Citation: Cole Sitron, F Ulrich Hartl 2025. In vitro coimmunoprecipitation between CHMP2B and alpha-synuclein. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rb66g1b/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: June 10, 2024
Last Modified: January 13, 2025
Protocol Integer ID: 103705
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-000282
Aligning Science Across Parkinson’s
Grant ID: ASAP- 024268
Abstract
This protocol details an in vitro coimmunoprecipitation between recombinant CHMP2B and different alpha-synuclein species - either monomers or fibrils. The goal of the experimental setup described herein is to assess which alpha-synuclein species CHMP2B preferentially binds.
Materials
• Recombinant alpha-synuclein A53T monomers and PFFs (5 mg/ml; prepared as in dx.doi.org/10.17504/protocols.io.btynnpve)
• Recombinant CHMP2B (1 mg/ml; prepared as in [https://www.protocols.io/view/purification-of-chmp2b-dg8c3zsw])
• ReagentRabbit anti-CHMP2B antibodyAbcamCatalog #ab33174
• CHMP2B buffer:
AB
Tris20 mM
pH7
NaCl150 mM
beta-mercaptoethanol2 mM
• PBS-T: 1X PBS pH 7.2 (Thermo Fisher Scientific cat. no. 20012068), 0.02% Tween-20ReagentPBS, pH 7.2Thermo Fisher ScientificCatalog #20012068
• 4X NuPAGE LDS Sample Buffer (Thermo Fisher Scientific cat. no. NP0007) supplemented with 5% 2-mercaptoethanolReagentNUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007
• Dynabeads Protein G for Immunoprecipitation (Invitrogen Cat. no. 10003D)ReagentDynabeads™ Protein G for ImmunoprecipitationThermo FisherCatalog #10003D
• Magnetic tube rack (e.g. Invitrogen cat. no. 12321D)ReagentMagnetic Tube Rack for 1.5mL or 15mL tubesContributed by usersCatalog #12321D
• Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific cat. no. A53225)ReagentPierce™ Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225
• Low bind 1.5 ml centrifuge tubes (Eppendorf cat. no. 0030108116)ReagentProtein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116

Sample Preparation
Sample Preparation
30m
30m
Thaw alpha-synuclein monomers, fibrils, and CHMP2B TemperatureOn ice .
Temperature
Centrifuge alpha-synuclein monomers and CHMP2B for Duration00:10:00 at Centrifigation16000 x g, 4°C .
10m
Centrifigation
Temperature
During the centrifugation, Vortex the Dynabeads vial until re-suspended.
Centrifigation
Mix
Pipette Amount20 µL of beads into an Eppendorf tube along with Amount500 µL PBS-T.
Pipetting
Place the tube in the magnetic stand to separate the beads from the solution.
Remove and discard the supernatant.
Add Amount2 µL of the anti-CHMP2B antibody along with Amount200 µL PBS-T into the tube.
Pipetting
Incubate at TemperatureRoom temperature , with rotation, for Duration00:20:00 to bind the antibody to beads.
20m
Incubation
Temperature
Once the centrifugation has ended, remove the supernatant and place the alpha-synuclein monomers and CHMP2B into new low bind tubes.
Centrifigation
Perform a Rapid BCA Gold Protein Assay on the alpha-synuclein monomers and CHMP2B to determine their concentrations.
Prepare 4 tubes:
5 µM monomers/CHMP2B:
AB
alpha-synuclein monomers18.4 µg
CHMP2B29.9 µg
PBS-T250 µl
5 µM PFFs/CHMP2B:

AB
alpha-synuclein PFFs18.4 µg
CHMP2B29.9 µg
PBS-T250 µl
5 µM monomers alone:

AB
alpha-synuclein monomers18.4 µg
CHMP2Bequivalent volume of CHMP2B buffer
PBS-T250 µl
5 µM PFFs alone:

AB
alpha-synuclein PFFs18.4 µg
CHMP2Bequivalent volume of CHMP2B buffer
PBS-T250 µl
Remove Amount12.5 µL of each tube and add to a new tube to run as an input fraction.
Dilute the input fraction with an additional Amount12.5 µL of PBS-T, then add Amount25 µL NuPAGE LDS sample buffer and denature for Duration00:10:00 at Temperature70 °C .
10m
Temperature
Immunoprecipitation
Immunoprecipitation
1h 5m
1h 5m
Place the tube with the beads on the magnetic rack and remove the supernatant.
Wash the beads one time with 1000 PBS-T.
Wash
Add the protein solutions to the tube with the beads and gently pipette to resuspend.
Pipetting
Incubate with rotation at TemperatureRoom temperature for Duration00:45:00 .
45m
Incubation
Temperature
Remove the tubes from the roller and place on the magnetic stand.
Remove the supernatant and keep in a separate tube to run as a flow-through fraction.
Take the tube with the beads off of the magnet. Add Amount1000 µL PBS-T and gently pipette to wash.
Pipetting
Wash
Put back on magnetic stand, and remove and discard the supernatant.
Repeat the wash step 5 times.
Wash
After your final wash, re-suspend the beads in Amount100 µL PBS-T and transfer to a clean tube.
Pipetting
Wash
Remove supernatant with magnetic rack.
Elute using Amount25 µL NuPAGE LDS sample buffer and Amount25 µL PBS-T and incubate for Duration00:10:00 at Temperature70 °C .
10m
Incubation
Pipetting
Temperature
Add Amount12.5 µL of the flow-through fraction to Amount12.5 µL PBS-T and Amount25 µL NuPAGE LDS sample buffer and denature for Duration00:10:00 at Temperature70 °C .
10m
Pipetting
Temperature
Remove the tubes containing the beads and place on the magnetic stand.
Remove the eluate and transfer to a new tube.