Mar 20, 2024

αS PFF Assembly Protocol

DOI
https://dx.doi.org/10.17504/protocols.io.kxygx3j3dg8j/v1
  • Scott Vermilyea1
  • 1University of Minnesota
  • Team Lee
Jane Balster
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DOI: https://dx.doi.org/10.17504/protocols.io.kxygx3j3dg8j/v1
Protocol Citation: Scott Vermilyea 2024. αS PFF Assembly Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx3j3dg8j/v1
License: This is an open access  protocol  distributed under the terms of the  Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 26, 2024
Last Modified: May 31, 2024
Protocol  Integer ID: 95780
Keywords: ASAPCRN, pff assembly protocol this protocol, αs pff assembly, pff assembly protocol, protocol this protocol detail, protocol detail, protocol
Abstract
This protocol details the αS PFF assembly.
Attachments
Icon for Generation of recombinant aS and PFF protocol.docx
Generation of recomb...
22KB
Aggregation
Remove monomer stock from -80 °C and thaw On ice .
Dilute stock to desired concentration (typically 5 µL ) to a final volume of 300 µL in autoclaved2 mL centrifuge tubes.
Set thermocycler for continuous shaking (1000 rpm, 37°C ).
Install 2 mL plate.
Run PBS and Protein samples. Take 2 µL aliquot at 0 hr time point. Run ThioT Assay.
Take 2 µL aliquots twice per day for up to 120:00:00 ( 72 hours - 96 hours may be sufficient).
5d
Check ThioT Fluorescence.
Aliquot and store at -80 °C .
ThioT Assay
Prepare ThioT stock mixture (8 µg ThioT in 10 µL milliQ H2O).
Prepare working ThioT mixture, 20 µL stock into 1 mL PBS.
Dispense 200 µL working solution into each 2 µL aliquot.
Dispense 200 µL per sample into 96-well, black, corning glass-bottomed plate.
Read Fluorescence Intensity (Ex 450 +/-10; Em 490 +/- 10).