αS PFF Assembly Protocol

Scott Vermilyea

Published: 2024-03-21 DOI: 10.17504/protocols.io.kxygx3j3dg8j/v1

ASAPCRN

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Abstract

This protocol details the αS PFF assembly.

Attachments

Generation of recombinant aS and PFF protocol.docx

Steps

Aggregation

1.

Remove monomer stock from -80°C and thaw On ice.

2.

Dilute stock to desired concentration (typically 5) to a final volume of 300µL in autoclaved2mLcentrifuge tubes.

3.

Set thermocycler for continuous shaking (1000rpm,37°C,0h 0m 0s ).

4.

Install 2mL plate.

5.

Run PBS and Protein samples. Take 2µL aliquot at 0 hr time point. Run ThioT Assay.

6.

Take 2µL aliquots twice per day for up to 120h 0m 0s ( 72 hours - 96 hours may be sufficient).

7.

Check ThioT Fluorescence.

8.

Aliquot and store at -80°C.

ThioT Assay

9.

Prepare ThioT stock mixture (8µg ThioT in 10µL milliQ H₂O).

10.

Prepare working ThioT mixture, 20µL stock into 1mL PBS.

11.

Dispense 200µL working solution into each 2µL aliquot.

12.

Dispense 200µL per sample into 96-well, black, corning glass-bottomed plate.

13.

Read Fluorescence Intensity (Ex 450 +/-10; Em 490 +/- 10).

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