Jul 04, 2024

Public workspaceProtein Aggregation Capture

This protocol is a draft, published without a DOI.
  • Mark Kinnin1
  • 1Queen's University Belfast
Mark Kinnin
Open access
Protocol Citation: Mark Kinnin 2024. Protein Aggregation Capture. protocols.io https://protocols.io/view/protein-aggregation-capture-dgpg3vjw
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
Abstract
PAC Adapted from Batth et al., 2019
Protocol materials
ReagentMagReSyn Hydroxyl BeadsResyn Biosciences
Step 1
ReagentAcetonitrile
Step 5
Reagent70% ACNThermo Scientific
In 2 steps
Reagent100% ACNThermo Scientific
Step 8
Reagent70% Ethanol
Step 9
ReagentTrypsinP212121Catalog #RP-T70010
Step 11
ReagentTrisP212121
Step 10
ReagentTFAThermo Scientific
In 2 steps
Equilibration
Equilibration
Add Amount5 µL of ReagentMagReSyn Hydroxyl BeadsResyn Biosciences to 1.5mL LoBind Eppendorf tube



Place tube on magnet and allow Microparticles to clear Duration00:00:10

10s
Critical
Remove storage solution and discard
Equilibrate Microparticles by adding Amount100 µL Reagent70% ACNThermo Scientific and mix by gentle agitation

Place tube on magnet and allow Microparticles to clear Duration00:00:10

10s
Critical
Remove supernatant and discard
Add Amount100 µL Reagent70% ACNThermo Scientific and mix by gentle agitation

Place tube on magnet and allow Microparticles to clear Duration00:00:10
10s
Critical
Remove supernatant and discard
Add Amount2 µL sample in triplicate

Add ReagentAcetonitrile Contributed by users to a concentration of 70% and mix once by pipette to create a uniform suspension

Allow to rest Duration00:10:00

10m
Incubation
Wash - DO NOT REMOVE FROM MAGNET
Wash - DO NOT REMOVE FROM MAGNET
10s
Place tube on magnet and allow Microparticles to clear Duration00:00:10

10s
Critical
Remove supernatant and discard
Add Amount1 mL Reagent100% ACNThermo Scientific and incubate for Duration00:00:10

10s
Remove supernatant
Add Amount1 mL Reagent70% Ethanol Contributed by users and incubate for Duration00:00:10

10s
Remove supernatant
Digestion
Digestion
10s
Remove tube from magnet and add Ph8.5 ReagentTrisP212121 and mix throughly
Add Amount5 µg ReagentTrypsinP212121Catalog #RP-T70010 and incubate for Duration04:00:00
4h
Quench to a final concentration Concentration1 % volume ReagentTFAThermo Scientific

Mix and place samples on magnet for Duration00:01:00 and transfer supernatant to tubes

1m
Wash beads with Amount50-100 µL Concentration1 % volume ReagentTFAThermo Scientific for Duration00:02:00 with continous mixing

2m
Remove supernatant and pool with eluate from Step 13
Centrifuge tubes Centrifigation20000 x g, 00:10:00

10m
Transfer supernatant to new tube and freeze down at Temperature-80 °C