Jul 04, 2024
Protein Aggregation Capture
This protocol is a draft, published without a DOI.
- Mark Kinnin1
- 1Queen's University Belfast
Protocol Citation: Mark Kinnin 2024. Protein Aggregation Capture. protocols.io https://dx.doi.org/
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: In development
We are still developing and optimizing this protocol
Created: July 04, 2024
Last Modified: July 04, 2024
Protocol Integer ID: 102856
Keywords: protein
Abstract
PAC Adapted from Batth et al., 2019
Protocol materials
MagReSyn Hydroxyl BeadsResyn Biosciences
Acetonitrile
70% ACNThermo Scientific
100% ACNThermo Scientific
70% Ethanol
TrypsinP212121Catalog #RP-T70010
TrisP212121
TFAThermo Scientific
Equilibration
Add 5 µL of MagReSyn Hydroxyl BeadsResyn Biosciences to 1.5mL LoBind Eppendorf tube
Place tube on magnet and allow Microparticles to clear 00:00:10
10s
Remove storage solution and discard
Equilibrate Microparticles by adding 100 µL 70% ACNThermo Scientific and mix by gentle agitation
Place tube on magnet and allow Microparticles to clear 00:00:10
10s
Remove supernatant and discard
Add 100 µL 70% ACNThermo Scientific and mix by gentle agitation
Place tube on magnet and allow Microparticles to clear 00:00:10
10s
Remove supernatant and discard
Add 2 µL sample in triplicate
Add Acetonitrile to a concentration of 70% and mix once by pipette to create a uniform suspension
Allow to rest 00:10:00
10m
Wash - DO NOT REMOVE FROM MAGNET
10s
Place tube on magnet and allow Microparticles to clear 00:00:10
10s
Remove supernatant and discard
Add 1 mL 100% ACNThermo Scientific and incubate for 00:00:10
10s
Remove supernatant
Add 1 mL 70% Ethanol and incubate for 00:00:10
10s
Remove supernatant
Digestion
10s
Remove tube from magnet and add 8.5 TrisP212121 and mix throughly
Add 5 µg TrypsinP212121Catalog #RP-T70010 and incubate for 04:00:00
4h
Quench to a final concentration 1 % volume TFAThermo Scientific
Mix and place samples on magnet for 00:01:00 and transfer supernatant to tubes
1m
Wash beads with 50-100 µL 1 % volume TFAThermo Scientific for 00:02:00 with continous mixing
2m
Remove supernatant and pool with eluate from Step 13
Centrifuge tubes 20000 x g, 00:10:00
10m
Transfer supernatant to new tube and freeze down at -80 °C