Nov 08, 2023

Public workspaceSingle-molecule pulldown for immunodetection of protein aggregates

DOI
dx.doi.org/10.17504/protocols.io.36wgq3bz3lk5/v1
  • 1University of Edinburgh
Rebecca Saleeb
Open access
DOI: dx.doi.org/10.17504/protocols.io.36wgq3bz3lk5/v1
Protocol Citation: Rebecca Saleeb, Craig Leighton, Ji-Eun Lee, Mathew Horrocks 2023. Single-molecule pulldown for immunodetection of protein aggregates. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq3bz3lk5/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: November 02, 2023
Last Modified: November 08, 2023
Protocol Integer ID: 90318
Funders Acknowledgement:
UCB Biopharma S.P.R.L.
MRC National Productivity Investment Fund studentship
MND Association Lady Edith Wolfson Junior Non-Clinical Fellowship
Grant ID: Saleeb/Oct22/980-799
Abstract
This protocol describe the procedures used to clean and passivate a coverglass surface, functionalise it with biotin and specifically immobilise proteins of interest for detection with single channel imaging (SiMPull), two-colour coincidence aggregate detection (STAPull) or ThT detection (SAVE imaging) on a TIRF microscope. This protocol supports published work (Saleeb et al., 2023).
Materials
Equipment:
  • Diener Electronic ZeptoOne Plasma Cleaner
  • Oxford Nanoimager

Consumbles:
  • Coverslips (Merck, C9056-1CS)
  • Ibidi 18-well gasket (Ibidi, 81818)
  • 0.02-micron syringe filters (Merck, WHA68092002)

Reagents:
  • Potassium hydroxide (Merck, 221473-500G)
  • Acetic acid (Merck, 33209-2.5L-M)
  • APTMS (Merck, 281778-5ML)
  • Methanol (Merck, 32213-2.5l-M)
  • mPEG-SVA (5000 Da, Laysan Bio)
  • Biotinylated mPEG-SVA (5000 Da, Laysan Bio)
  • Sodium hydrogen carbonate (Fisher Scientific, 10020510)
  • Streptavidin (ThermoFisher Scientific, 21122)
  • Capture antibody, PS129 anti-aSynuclein (Abcam, ab209422) biotinylated by kit (Pierce, 90407)
  • Detection antibody, 1:1 mix of PS129 anti-aSynuclein labelled with AF488 or labelled with AF647 by kit (ThermoFisher, A20181/6)
  • Thioflavin T (Merck, 596200-500MG)
Coverslip passivation and functionalisation
Coverslip passivation and functionalisation
Treat coverslips with argon plasma for 45 mins
Immerse coverslips in 0.22 micron-filtered 1M potassium hydroxide for 20mins
Rinse coverslips in ultrapure deionised water and immerse in APTMS solution (prepared as 3.75 mL glacial acetic acid, 1 mL APTMS, 75 mL methanol) and incubate covered for 20 mins at RT.
Rinse coverslips sequentially in methanol and ultrapure deionised water and use fast flowing argon to dry slides rapidly. Inspect slide to ensure visually clean.
Attach 18-well Ibidi sticky-slide chamber to coverslip, apply pressure to ensure fully adhered.
Dispense 50 uL mPEG solution per well (95mg/mL mPEG-SVA, 5 mg/mL biotin-PEG, 0.1M sodium hydrogen carbonate), parafilm seal chamber and incubate covered overnight at RT.
Sample pull-down and labelling
Sample pull-down and labelling
Empty PEG solution by inversion and wash wells with ultrapure deionised water x3
Dry wells thoroughly using fast-flowing argon gas
Apply 50 uL 0.2 mg/mL streptavidin prepared in 0.02-micron filtered T50 buffer (10 mM Tris pH 8.0 supplemented with 50 mM sodium chloride) per well and incubate covered for 10mins at RT
Wash wells with 0.02-micron filtered T50 buffer x3
Apply 50 uL of 100 nM biotinylated capture antibody and incubate covered for 20 mins at RT
Wash wells with 0.02-micron filtered PBS x3
Apply 50 uL neat CSF per well and incubate covered for 24 hours at RT
Wash wells with 0.02-micron filtered PBS x3

NB. CSF is biohazardous, aspirate to remove liquid waste instead of slide inversion
Apply 50 uL 2nM fluorophore-labelled detection antibody and incubate covered for 3 hours at RT
Wash wells with 0.02-micron filtered PBS x3
Apply 50 uL PBS per well and image immediately
Thioflavin T preparation
Thioflavin T preparation
Prepare approx. 4 mM stock of ThT in 100% ethanol and vortex extensively (approx. 1 hour)
Prepare approx. 200 uM ThT dilution in PBS, vortex thoroughly (approx. 20 mins) and filter through 0.02-micron filter.
Measure concentration of ThT preparation using a DeNovix spectrophotometer (extinction coefficient 36,000 M-1 cm-1 at 412 nm)
Prepare a 10 uM working stock in 0.02-micron filtered PBS
Imaging
Imaging
Acquire an 8 x 8 grid of 200-micron spaced fields of view per well by total internal reflection fluorescence microscopy using the ONI Nanoimager with 100x/1.4 oil immersion objective lens. Samples are sequentially excited at 638nm and 488 nm, 20 frames captured per field in each channel at 20 frames s-1.
On completion of imaging one well, return to position 0 (based on metadata values), cautiously apply 50 uL 10 uM ThT and re-image all fields with 405nm excitation.