Home›Cell Biology›Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
Cell BiologyJoVE (Open Access)Citable · DOI
Generation of Alpha-Synuclein Preformed Fibrils from Monomers and Use In Vivo
DOI: 10.3791/59758-v
What you'll learn
✓Generate alpha-synuclein preformed fibrils from monomeric protein
✓Perform quality control analysis of fibril preparations
✓Prepare stereotactic injection needles for in vivo delivery
✓Administer preformed fibrils to animal models
Protocol
Biopharma Insights The goal of this article is to outline the steps required for the generation of fibrils from monomeric alpha-synuclein, subsequent quality control, and use of the preformed fibrils in vivo.
Difficulty
advanced
Total time
~3-5 days (fibril generation and quality control); ~2 hours per animal (stereotactic surgery)
Model organism
Mouse (unspecified strain)
Biosafety
BSL-1
Steps
1
Form alpha-synuclein preformed fibrils from monomers
Incubate monomeric alpha-synuclein under conditions that promote self-assembly into fibrillar structures. Monitor fibril formation over time.
▶ 01:13
2
Sonicate alpha-synuclein preformed fibrils
Fragment the generated fibrils via sonication to produce smaller, uniform fibril seeds suitable for injection. Confirm size reduction.
▶ 02:20
3
Prepare custom glass needle syringes for injection
Pull and polish glass capillaries to create fine-gauge needles compatible with stereotactic apparatus for precise brain injection.