Mar 29, 2026

Public workspaceMouse alpha-synuclein preformed fibril preparation

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkyd1xg5r/v1
  • Sayan Dutta1
  • 1California Institute of Technology
Sayan Dutta
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkyd1xg5r/v1
Protocol Citation: Sayan Dutta 2026. Mouse alpha-synuclein preformed fibril preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkyd1xg5r/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: September 09, 2025
Last Modified: March 29, 2026
Protocol Integer ID: 226797
Keywords: PFF, Parkinson's disease, alpha-synuclein, fibril for parkinson, synuclein, fibril preparation purification, fibril, preparation of mouse alpha, parkinson, disease modeling in rodent, mouse alpha
Abstract
Purification and preparation of mouse alpha-synuclein preformed fibril for Parkinson's disease modeling in rodents.
Materials
Materials & reagents (examples / catalog numbers from your text)
  • E. coli BL21 (DE3) transformed with pT7-7 encoding mouse α-synuclein
  • LB medium + ampicillin (100 µg/mL)
  • IPTG (isopropyl β-D-1-thiogalactopyranoside)
  • Lysis buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 mg/mL lysozyme
  • Streptomycin sulfate (0.1% w/v)
  • Ammonium sulfate (for 30% and 50% saturation)
  • 10 mM Tris-HCl pH 7.4, 1 mM EDTA (for resuspension and SEC/ion exchange buffers)
  • PBS (10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, 2.7 mM KCl, 137 mM NaCl, pH 7.4)
  • Pierce high-capacity endotoxin removal resin (Thermo #88277)
  • 0.22 µm syringe filter (Corning #8160)
  • DPBS for fibril resuspension
  • Guanidine hydrochloride 8 M (for fibril dissociation QC)
  • Consumables: French press tubing or spare parts, sterile 1.5 mL microtubes, dialysis tubing, sonication tubes (e.g., Active Motif #53071)

Equipment

  • Incubator shaker (37°C)
  • Centrifuge capable of 13,500 × g, refrigerated
  • French press / cell disruptor (or other high-pressure lysis device)
  • Thermomixer (with 1000 rpm capability)
  • Size exclusion column: HiLoad 16/600 Superdex 200 pg (Cytiva)
  • Anion exchange column: HiPrep Q HP 16/10 or DEAE (Cytiva)
  • Nanodrop or spectrophotometer (A₂₈₀ measurement)
  • Cup-horn sonicator (Qsonica q700) and chilled water/ice bath
  • SDS-PAGE setup and Coomassie stain
Troubleshooting
Recombinant α-synuclein expression
Inoculate BL21(DE3) + pT7-7 (mouse α-syn) into LB + ampicillin (100 µg/mL). Grow at 37°C with shaking until OD₆₀₀ = 0.5–0.6.
Induce expression with IPTG to a final concentration of 1 mM. Continue incubation at 37°C for 4 h.
Harvest cells by centrifugation (e.g., 5,000 × g, 10 min, 4°C). Proceed immediately to lysis or freeze pellets at −80°C.
Cell lysis and salting out
Resuspend cell pellet in lysis buffer: 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.25 mg/mL lysozyme (use ~5–10 mL buffer per g wet cell pellet). Incubate briefly on ice (5–15 min) to allow lysozyme action.
Lyse cells using a French press (≥1000 psi) or equivalent. Keep lysate cold.
Add streptomycin sulfate to 0.1% (w/v) to precipitate nucleic acids; mix and incubate briefly (a few minutes).
Clarify lysate by centrifugation (e.g., 15,000 × g, 20–30 min, 4°C). Collect supernatant.
Perform ammonium sulfate precipitation sequentially at 4°C: first to 30% saturation, remove pellet (optional), then increase to 50% saturation and collect pellet containing α-syn. (Adjust volumes/salt amounts per standard ammonium sulfate tables.)
Resuspend the 50% pellet in 10 mM Tris-HCl pH 7.4.
Heat the resuspended sample at 95°C for 15 min (boiling step) to denature many contaminants. Cool on ice.
Centrifuge at 13,500 × g, 20 min, 4°C to pellet denatured proteins. Retain the supernatant (contains soluble α-syn).
Chromatography and Dialysis
Size exclusion chromatography (SEC): Load supernatant on HiLoad 16/600 Superdex 200 pg, elute in 10 mM Tris-HCl pH 7.4. Collect fractions.
Anion exchange: Pool α-syn-enriched SEC fractions (monitor by SDS-PAGE), then run on HiPrep Q HP 16/10 or DEAE in 10 mM Tris-HCl pH 7.4 + 1 mM EDTA. Elute with a linear NaCl gradient from 25 mM → 1 M NaCl. Collect fractions.
Analyze fractions by SDS-PAGE (Coomassie) and pool those with α-syn (target purity ≈ 95%).
Dialyze pooled fractions against PBS (pH 7.4) to exchange buffer.
Endotoxin removal
Remove endotoxin using Pierce high-capacity endotoxin removal resin per manufacturer’s instructions; aim for ≤0.018 EU/µg protein (verify with LAL or equivalent endotoxin assay).
Determine protein concentration (see QC below). Aliquot and store at −80°C.
Expected: final purity ~95% (SDS-PAGE).
Fibril formation and sonication
Filter monomeric α-syn solution (prepare at 5 mg/mL) through a 0.22 µm filter into sterile 1.5 mL microcentrifuge tubes. Example: 500 µL of 5 mg/mL.
Incubate at 37°C with continuous agitation at 1,000 rpm in a Thermomixer for 7 days to form fibrils. Use sterile conditions.
After 7 days, pellet fibrils by centrifugation at 13,000 × g, 10 min. Carefully remove and discard supernatant. Resuspend pellet in 250 µL DPBS.
Take a small aliquot (5 µL) of fibril suspension and incubate with 8 M guanidine-HCl (5 µL) at 22°C for 1 h to dissociate fibrils into monomers for concentration measurement.
Measure A₂₈₀ on a Nanodrop / spectrophotometer. Use extinction coefficient ε₂₈₀ = 7450 M⁻¹ cm⁻¹ to calculate molar concentration (A / ε), then convert to mg/mL using the molecular weight (use literature value for mouse α-syn).
Formula: Molarity (M) = A₂₈₀ / 7450. Mass concentration (g/L) = M × MW. Convert to mg/mL as needed. Note: Adjust for guanidine-HCl dilution step.
Store fibrils as 25 µL aliquots at 5 mg/mL at −80°C.
Thaw a 25 µL aliquot on ice when ready to use. Transfer to an ethanol-sterilized sonication tube (e.g., Active Motif #53071).
Sonicate in a cup-horn sonicator (Qsonica q700) at 30% amplitude (~100 W/s) using a cycle of 3 s ON / 2 s OFF, for a total ‘on’ time of 15 min (i.e., multiple cycles). Maintain water bath temperature between 5–15°C (use ice or chilled circulating bath).
After sonication, keep samples at room temperature and use promptly for injections or downstream assays.
Protocol references
Zhang, H., Griggs, A., Rochet, J.C. & Stanciu, L.A. In vitro study of alpha-synuclein protofibrils by cryo-EM suggests a Cu(2+)-dependent aggregation pathway. Biophys J 1 4, 2706-13 (2013).
Volpicelli-Daley, L.A., Luk, K.C. & Lee, V.M. Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha synuclein to Lewy body and Lewy neurite-like aggregates. Nat Protoc 9, 2135-46 (2014).

Stoyka, L.E. et al. Behavioral defects associated with amygdala and cortical dysfunction in mice with seeded alpha-synuclein inclusions. Neurobiol Dis 134, 104708 (2020).