Jan 10, 2026
α-Synuclein binding assay to isolated synaptic vesicles
- Akio Mori1,
- Robert Edwards1
- 1University of California San Francisco
Protocol Citation: Akio Mori, Robert Edwards 2026. α-Synuclein binding assay to isolated synaptic vesicles. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqp8mxvk5/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: November 20, 2025
Last Modified: January 10, 2026
Protocol Integer ID: 232990
Keywords: Synaptic vesicle, Pull-down assay, α-synuclein, assay to isolated synaptic vesicle, isolated synaptic vesicle, synaptic vesicle, synuclein, syn, binding assay, mouse brain
Abstract
The binding of α-synuclein (α-syn) to synaptic vesicles was assessed by pull-down using C-terminally His-tagged α-syn and Co Dynabeads (10103D, Invitrogen). Synaptic vesicles (SVs) were isolated from mouse brain (LP2 fraction), incubated with recombinant α-syn, and the bound fractions were analyzed by immunoblotting.
Materials
- Co Dynabeads (10103D, Invitrogen)
- Synaptic vesicles (SVs; LP2 fraction) purified from mouse brain
- Reaction buffer: 150 mM KCl, 1 mM MgCl₂, 20 mM HEPES, pH 7.4
- BSA (A8806, Sigma)
- SHT buffer: 0.3 M sucrose, 1 mM Mg-EGTA, 10 mM HEPES-Tris, pH 7.4
- Recombinant His-tagged α-synuclein
- Wash buffer: 150 mM KCl, 1 mM MgCl₂, 5 mM imidazole, 20 mM HEPES, pH 7.4
- Elution buffer: 150 mM KCl, 1 mM MgCl₂, 200 mM imidazole, 20 mM HEPES, pH 7.4
- Antibody: synaptophysin (mouse monoclonal, 1:1,000; S5768, Sigma-Aldrich)
Troubleshooting
Beads Preparation
12.5 μL per reaction of Co Dynabeads (10103D, Invitrogen) are transferred to a 1.5 mL low-adhesion microcentrifuge tube.
Wash the beads three times with 200 μL reaction buffer (150 mM KCl, 1 mM MgCl2, 20 mM HEPES, pH 7.4 ).
Pre-block Co Dynabeads with 1% BSA in reaction buffer for 30 min at room temperature with gentle rotation.
- This step reduces nonspecific binding of SVs to the beads.
Wash the beads three times with reaction buffer.
Binding reaction
Resuspend the LP2 fraction in SHT buffer (1mg/mL)
Incubate purified recombinant His-tagged α-syn (0–25 μg) with 25 μg of LP2 in reaction buffer (total volume 100 μL) for 3 h at room temperature with gentle agitation. Collect a mixture as input for Western blotting analysis.
Add the mixture to the pre-blocked beads and incubate for 15 min at room temperature with gentle rotation.
Washing and elution
Place tubes on a magnetic stand and collect the beads. Wash three times with 1 mL wash buffer.
Elute bound proteins by incubating beads in 25 μL elution buffer for 15 min at room temperature with gentle rotation.
Spin briefly at 1,000 × g for 30 s.
Place on a magnetic stand and collect the eluted fraction.
Analysis
Analyze pull-down efficiency by immunoblotting.
Quantify band intensities using ImageJ.
Normalize synaptophysin levels in the elution fraction to the input.