Pre-Formed Fibrils, WT α-Synuclein (α-syn-WT PFFs)

Pre-formed fibrils (PFFs) of wild type (WT) alpha-synuclein.

Alpha-synuclein (α-synuclein) is an intrinsically disordered protein of unknown function that is linked to both familial and sporadic cases of Parkinson's disease, where it forms the major constituent of the pathological Lewy body aggregates. These fibrils can be used for disease modelling since similar PFFs have been shown to seed endogenous α-synuclein inclusions in healthy cells.

From the laboratory of Scott D. Ryan, PhD, University of Guelph.

Catalog Number Product Size AVAILABILITY Price Qty
EGP018
Pre-Formed Fibrils, WT α-Synuclein (α-syn-WT PFFs)
1mg 1-2 weeks
Regular Price:$225.00
Specifications

Product Type: Protein
Name: pre-formed fibrils (PFFs) of WT alpha-synuclein
Alternative Name(s): alpha-syn-WT PFFs
Accession ID: P37840
Source: Human, expressed in E. coli BL21 (DE3) CP+
Amino Acid Sequence: MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA
Fusion Tag(s): None
Purity: >95 %, purified by ion-exchange chromatography followed by reverse phase chromatography
Buffer: Dulbeccos Phosphate Buffered Saline, Sigma D8537
Comments: A sedimentation assay was completed to confirm PFF formation
Storage: -80C
Shipped: Dry Ice

Provider
From the laboratory of Scott D. Ryan, PhD, University of Guelph.
Comments

Preparing PFFs (Pre-Formed Fibrils) for Treatment of Primary Neurons

SAFETY: Wear gloves, disposable sleeves, and a second pair of gloves when handling PFFs. Dispose of pipette tips and microfuge tubes in a container of 1% SDS. Wear protective headphones while sonicating fibrils, and decontaminate probe and spills with 1% SDS.

Preparation:

  1. Obtain stock PFFs from -80 freezer (stocks contain 10uL of 5mg/mL fibrils, and contain either WT or mutant α-synuclein; WT is used for most applications).
  2. Place PBS, 1% SDS and a P1000 in TC hood. Don sleeves and second pair of gloves.
  3. Pipette PBS into PFF aliquot depending on desired concentration (commonly 490uL is added to give a concentration of 0.1mg/mL) and place used tip in SDS.
  4. Remove outer gloves when leaving hood.

 

Sonication:

  1. Attach 1/8Ó microtip to sonicator (if not previously attached) and use wrench to tighten
  2. Clean the probe and the door of the enclosure with 70% EtOH.
  3. Set the sonicator to the following settings: 20% amplitude; 30 seconds; 1 second on, 1 second off
  4. Don a second pair of gloves.
  5. Place fibril tube in a tube rack in the sonicator enclosure
  6. Adjust the stand height such that the probe tip is submerged in the solution (about halfway between the solution surface and tube bottom), and adjust the rack so that the probe is in the middle of the tube.
  7. Remover outer gloves, close enclosure, don protective headphones, make sure Do Not Enter sign is on door, and press start.
  8. If bubbles form or the probe loosens during sonication, stop the sonication, don a second pair of gloves, and adjust as necessary.
  9. After sonication, don a second pair of gloves, remove and close the PFF tube, which is now ready for use.
  10. Clean the probe and anything the PFFs touched with 1% SDS. Clean again with 70% EtOH.

 

In cell culture, these sonicated PFFs are typically used at 1% vol/vol (or 10uL/mL of media).

References
  1. Ryan, T., Bamm, V.V., Stykel, M.G., Coackley, C.L., Humphries, K.M., Jamieson-Williams, R., Ambasudhan, R., Mosser, D.D., Lipton, S.A., Harauz, G., et al. (2018). Cardiolipin exposure on the outer mitochondrial membrane modulates α-synuclein. Nat. Commun 9, 1-17. doi:10.1038/s41467-018-03241-9.
  2. Stykel, M.G., Humphries, K., Kirby, M.P., Czaniecki, C., Wang, T., Ryan, T., Bamm, V., Ryan, S.D. (2018). Nitration of microtubules blocks axonal mitochondrial transport in a human pluripotent stem cell model of Parkinsons disease. Faseb. J 32, 5350-5364. doi:10.1096/fj.201700759RR.

If you publish research with this product, please let us know so we can cite your paper.

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