TITLE: Preparation of fibrils and Quality control
AUTHORS: The Michael J Fox Foundation Pff Standardization Consortium

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a consensus protocol developed through discussions with Laura Volpicelli-Daley, Caryl Sortwell, Kelvin Luk, Lindsey Gottler, and Virginia Lee. This protocol is intended for research purposes only, using specially-formulated monomeric alpha-synuclein protein available for purchase at Proteos, Inc as the result of efforts by The Michael J. Fox Foundation (MJFF). Each batch of the “Alpha-Synuclein Monomer Protein for Making Pre- Formed Fibrils” has undergone internal purification and quality control at Proteos in addition to external validation to confirm successful generation of pathogenic aSyn PFFs. See Reference section for methods and results from application of alpha-synuclein pre-formed fibrils (aSyn PFFs) in primary neuron cultures in vitro or in mice in vivo. This protocol is referenced in the Polinski et al 2018 paper entitled "Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson's Disease in Rodents" (doi: 10.3233/JPD-171248).</div></div>

[STEPS]
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Thaw aliquot of “Alpha-Synuclein Monomer Protein for Making PreFormed Fibrils” or other recombinant aSyn monomer .
on ice
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Centrifuge at  for  in benchtop centrifuge at highest speed (-).
4 °C
Centrifuge: 12000 34
Centrifuge: 15000 34
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Retain only the supernatant with a pipette, avoiding any aSyn that may have pelleted. Determine the protein concentration of the sample.METHOD 1 (most recommended): Measure protein by A280 on a nanodrop device. Use Beer’s law to measure concentration (ε for synuclein = voo5960 M−1 cm−1 for human synuclein and 7450 M−1 cm−1 for mouse synuclein).METHOD 2 (less recommended): Perform BCA protein assay on this material to determine final protein concentration. We recommend performing the assay at 3 dilutions of protein (in triplicate for each dilution) to obtain accurate measurements.
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Assemble the pre-formed fibrils (PFFs) in  microcentrifuge tubes by diluting the monomeric protein into PBS for a final concentration of .
1.5 mL
EXAMPLE: If the protein concentration is  , add   protein to   PBS in a   tube. If the protein concentration is   (in   Tris,   NaCl,  ), add   of a   phosphate,   NaCl solution to   of the monomeric aSyn sample. This will result in a final buffer formulation of ~  NaCl, ~  Tris, and ~  phosphate with a pH of 7.2-7.6.NOTE: Because activity can decline over time when the PFFs undergo freeze-thaw cycles, we recommend that reactions not exceed   per tube.
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Vortex tubes at high speed for  to mix contents.
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Place microcentrifuge lid lock on lid of tube to prevent opening of lid. Label and date tube.
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Place in orbital shaker (e.g. Eppendorf Thermomixer R) at .
37 °C
NOTE: It is recommended to place the entire shaker in a 37ºC incubator since most shakers do not heat the top of the tube, resulting in condensation.
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Shake for  at . Solution should turn turbid during this period.
Centrifuge: 1000 33
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Pipet  aliquots in small tubes using gel loading pipet tips.
25 µl
?. [Step 1. Preparation of fibrils. (Timing ~30 min; 7 days for fibril formation)]
Freeze aliquots on dry ice and store at  or store aliquots at .
-80 °C
-80 Room temperature
?. [Step 2. Quality control to verify fibril formation]
Thaw an aliquot at  and perform Thioflavin T assay toconfirm presence of amyloid fibrils:
-80 Room temperature
?. [Step 2. Quality control to verify fibril formation]
Dilute  Thioflavin T stock in PBS to  final concentration (1:40 dilution).
?. [Step 2. Quality control to verify fibril formation]
Pipet  of the  Thioflavin T per well of 384 well plate.
95 µl
?. [Step 2. Quality control to verify fibril formation]
Pipet PFFs up and down to mix, add  to wells with Thioflavin T.
2.5 µl
?. [Step 2. Quality control to verify fibril formation]
For controls, include  PBS alone and  monomeric aSyn.
2.5 µl
2.5 µl
?. [Step 2. Quality control to verify fibril formation]
Incubate at  for  to .
-80 Room temperature
?. [Step 2. Quality control to verify fibril formation]
Read plate (excitation , emission ).
450
50
The presence of amyloid-like fibrils typically result in readings that are 20-100 fold higher than samples containing monomeric protein only for the human aSyn.
?. [Step 2. Quality control to verify fibril formation]
12. The presence of fibrils can also be assessed by sedimentation.
?. [Step 2. Quality control to verify fibril formation]
Dilute  of  PFFs in  PBS.
2 µl
20 µl
?. [Step 2. Quality control to verify fibril formation]
Spin in ultracentrifuge (e.g. TLA-100) at  for  at .
Centrifuge: 100000 34
25 °C
?. [Step 2. Quality control to verify fibril formation]
Remove supernatant, dilute in 5X Laemlli buffer.
?. [Step 2. Quality control to verify fibril formation]
Add  PBS to pellet, pipet up and down several times until resuspended, dilute in 5X Laemlli buffer.
20 µl
?. [Step 2. Quality control to verify fibril formation]
Boil samples at  for .
95 °C
?. [Step 2. Quality control to verify fibril formation]
Run equal volumes of supernatant and pellet fractions on 15% polyacrylamide gel.
?. [Step 2. Quality control to verify fibril formation]
Stain with coomassie brilliant blue to visualize bands.
Properly generated PFFs should result in equal amounts of protein in the supernatant and pellet fractions or greater amounts of protein in the pellet versus the supernatant fraction. If more protein is in the supernatant versus the pellet, PFF formation has been suboptimal.
?. [Step 2. Quality control to verify fibril formation]
Keep records of results of Thioflavin T and sedimentation assay to make batch to batch comparisons.
NOTE: Thioflavin T and sedimentation assays are basic biochemical analyses to verify general fibril formation. More quality control is recommended when testing a new protocol for generating PFFs or generating PFFs for the first time in one’s lab. More quality control is also recommended before commencing a long-term in vivo study with aSyn PFFs. For examples for additional recommended quality control experiments, see the table in Page 1.