Jan 13, 2025
In vitro coimmunoprecipitation between CHMP2B and alpha-synuclein
- Cole Sitron1,
- F Ulrich Hartl1
- 1Max Planck Institute of Biochemistry
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, synuclein species chmp2b, synuclein species, synuclein this protocol detail, coimmunoprecipitation between recombinant chmp2b, synuclein, coimmunoprecipitation between chmp2b, recombinant chmp2b, chmp2b, coimmunoprecipitation
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])
• Rabbit anti-CHMP2B antibodyAbcamCatalog #ab33174
• CHMP2B buffer:
| A | B | |
| Tris | 20 mM | |
| pH | 7 | |
| NaCl | 150 mM | |
| beta-mercaptoethanol | 2 mM |
• PBS-T: 1X PBS pH 7.2 (Thermo Fisher Scientific cat. no. 20012068), 0.02% Tween-20PBS, pH 7.2Thermo Fisher ScientificCatalog #20012068
• 4X NuPAGE LDS Sample Buffer (Thermo Fisher Scientific cat. no. NP0007) supplemented with 5% 2-mercaptoethanolNUPAGE LDS sample buffer (4x)Thermo Fisher ScientificCatalog #NP0007
• Dynabeads Protein G for Immunoprecipitation (Invitrogen Cat. no. 10003D)Dynabeads™ Protein G for ImmunoprecipitationThermo FisherCatalog #10003D
• Magnetic tube rack (e.g. Invitrogen cat. no. 12321D)Magnetic Tube Rack for 1.5mL or 15mL tubesCatalog #12321D
• Pierce Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific cat. no. A53225)Pierce™ Rapid Gold BCA Protein Assay KitThermo FisherCatalog #A53225
• Low bind 1.5 ml centrifuge tubes (Eppendorf cat. no. 0030108116)Protein LoBind Tubes, 1.5 mLEppendorfCatalog #0030108116
Troubleshooting
Sample Preparation
30m
Thaw alpha-synuclein monomers, fibrils, and CHMP2B On ice .
Centrifuge alpha-synuclein monomers and CHMP2B for 00:10:00 at 16000 x g, 4°C .
10m
During the centrifugation, Vortex the Dynabeads vial until re-suspended.
Pipette 20 µL of beads into an Eppendorf tube along with 500 µL PBS-T.
Place the tube in the magnetic stand to separate the beads from the solution.
Remove and discard the supernatant.
Add 2 µL of the anti-CHMP2B antibody along with 200 µL PBS-T into the tube.
Incubate at Room temperature , with rotation, for 00:20:00 to bind the antibody to beads.
20m
Once the centrifugation has ended, remove the supernatant and place the alpha-synuclein monomers and CHMP2B into new low bind tubes.
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:
| A | B | |
| alpha-synuclein monomers | 18.4 µg | |
| CHMP2B | 29.9 µg | |
| PBS-T | 250 µl |
5 µM PFFs/CHMP2B:
| A | B | |
| alpha-synuclein PFFs | 18.4 µg | |
| CHMP2B | 29.9 µg | |
| PBS-T | 250 µl |
5 µM monomers alone:
| A | B | |
| alpha-synuclein monomers | 18.4 µg | |
| CHMP2B | equivalent volume of CHMP2B buffer | |
| PBS-T | 250 µl |
5 µM PFFs alone:
| A | B | |
| alpha-synuclein PFFs | 18.4 µg | |
| CHMP2B | equivalent volume of CHMP2B buffer | |
| PBS-T | 250 µl |
Remove 12.5 µL of each tube and add to a new tube to run as an input fraction.
Dilute the input fraction with an additional 12.5 µL of PBS-T, then add 25 µL NuPAGE LDS sample buffer and denature for 00:10:00 at 70 °C .
10m
Immunoprecipitation
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.
Add the protein solutions to the tube with the beads and gently pipette to resuspend.
Incubate with rotation at Room temperature for 00:45:00 .
45m
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 1000 µL PBS-T and gently pipette to wash.
Put back on magnetic stand, and remove and discard the supernatant.
Repeat the wash step 5 times.
After your final wash, re-suspend the beads in 100 µL PBS-T and transfer to a clean tube.
Remove supernatant with magnetic rack.
Elute using 25 µL NuPAGE LDS sample buffer and 25 µL PBS-T and incubate for 00:10:00 at 70 °C .
10m
Add 12.5 µL of the flow-through fraction to 12.5 µL PBS-T and 25 µL NuPAGE LDS sample buffer and denature for 00:10:00 at 70 °C .
10m
Remove the tubes containing the beads and place on the magnetic stand.
Remove the eluate and transfer to a new tube.