αS PFF Assembly Protocol
Scott Vermilyea
Abstract
This protocol details the αS PFF assembly.
Attachments
Generation of recombinant aS and PFF protocol.docx
Steps
Aggregation
Remove monomer stock from -80°C and thaw On ice.
Dilute stock to desired concentration (typically 5) to a final volume of 300µL in autoclaved2mLcentrifuge tubes.
Set thermocycler for continuous shaking (1000rpm,37°C,0h 0m 0s ).
Install 2mL plate.
Run PBS and Protein samples. Take 2µL aliquot at 0 hr time point. Run ThioT Assay.
Take 2µL aliquots twice per day for up to 120h 0m 0s ( 72 hours - 96 hours may be sufficient).
Check ThioT Fluorescence.
Aliquot and store at -80°C.
ThioT Assay
Prepare ThioT stock mixture (8µg ThioT in 10µL milliQ H2O).
Prepare working ThioT mixture, 20µL stock into 1mL PBS.
Dispense 200µL working solution into each 2µL aliquot.
Dispense 200µL per sample into 96-well, black, corning glass-bottomed plate.
Read Fluorescence Intensity (Ex 450 +/-10; Em 490 +/- 10).
