Jun 15, 2026

Microscopy-based bead assay

DOI
https://dx.doi.org/10.17504/protocols.io.e6nvw4kr7lmk/v1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.e6nvw4kr7lmk/v1
Protocol Citation: Elisabeth Holzer, Justyna Sawa-Makarska 2026. Microscopy-based bead assay. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4kr7lmk/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
-
Created: July 31, 2025
Last Modified: June 15, 2026
Protocol  Integer ID: 223986
Keywords: ASAPCRN, agarose bead immobilization, multiple bead, atg8 lipidation machinery, bait protein, liposome, based bead, lipid transfer experiment, tagged bait protein, based assay, protein interaction, assay, enzymatic activity, binding experiment, recombinant atg2a, involving recombinant atg2a
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
DOC Fellowship (Austrian Academy of Sciences)
Abstract
Summary: This protocol describes multiple bead-based assays designed to assess protein interactions and enzymatic activity. Assays include binding experiments with GFP- or GST-tagged bait proteins, activity assays with PI3KC3-C1 and ATG8 lipidation machinery, and tethering or lipid transfer experiments involving recombinant ATG2A and liposomes. All assays utilize agarose bead immobilization followed by imaging.
Materials
  • ChromoTek GFP-Trap® AgaroseProteintechCatalog # gta-20
  • Glutathione Sepharose 4B beads (GE Healthcare, Cat# 17075605)
  • ATG9A vesicles or proteoliposomes (PLs)
  • Recombinant proteins: ATG2A, PI3KC3-C1, mCherry-2xFYVE, mCherry-PI3KC3-C1, ATG2A-mCherry, GABARAP variants, ATG7, ATG3, E3 complex (ATG5-ATG12-ATG16L1)
  • Donor liposomes and Rhodamine-labeled liposomes

Equipment
Glass-bottom 384-well microplates
NAME
SENSOPLATE
BRAND
781892
SKU
https://shop.gbo.com/en/india/products/bioscience/microplates/sensoplate-glass-bottom-plates/384-well-sensoplate/781892.html
LINK

  • Confocal microscope (e.g. Zeiss LSM 700)
  • Buffers (see below)

Buffers:

  • Elution Base Buffer (EBB):

AB
HEPES pH 7.520 mM
mM NaCl150 mM
cOmplete EDTA-free protease inhibitor cocktail (Roche)1x
β-glycerophosphate20 mM
sodium orthovanadate1 mM
NaF1 mM
SEC150:
AB
HEPES pH 7.525 mM
NaCl150 mM
DTT1 mM
SEC500:
AB
HEPES pH 7.525 mM
NaCl500 mM
DTT1 mM
Cofactor mix:
AB
ATP0.5 mM
MgCl₂0.5 mM
MnCl₂2 mM
EGTA1 mM

Binding assays using ATG9A vesicles
10h
Equilibrate 10 µL of GFP-Trap agarose beads with water and twice with EBB.

Incubate freshly purified ATG9A vesicles (from 5x 15-cm plates) or MOCK controls (from wild-type HAP1 cells) with beads at 4 °C for 05:00:00 with gentle rotation.
5h
Store Overnight at 4 °C .
5h
Wash beads twice with 200 µL SEC150 and resuspend in 5 µL –10 µL SEC150.
Binding assays using ATG9A proteoliposomes
Equilibrate 10 µL GFP-Trap beads with water and twice with SEC150.
Incubate 500 µL of PLs with beads at 4 °C .
After binding, wash beads with SEC150 before downstream applications.
Testing the interaction of PI3KC3-C1 or ATG2A with ATG9A vesicles
30m
Pipette 20 µL prey protein (0.1 micromolar (µM) mCherry-PI3KC3-C1 or ATG2A-mCherry) into each well.
Add 1 µL of bead suspension (coated with ATG9A vesicles or MOCK control) per well.
Incubate 00:30:00 at Room temperature prior to imaging.
30m
PI3KC3-C1 activity assay
40m
Incubate vesicle/PL-coated GFP-Trap beads with 0.03 micromolar (µM) PI3KC3-C1 for 00:30:00 .
30m
Add 0.03 micromolar (µM) mCherry-2xFYVE and incubate 00:10:00 .
10m
Add cofactors to initiate PI(3)P production.
Reaction volume: 20 µL per well.
Lipid transfer assay
8h 5m
Incubate 125 µL ATG9A PLs with 10 micromolar (µM) ATG2A and 6.25 µL donor liposomes (0.5 mg/mL ) Overnight at 4 °C .
3h
Bind to 5 µL GFP-Trap beads for 05:00:00 .
5h
Wash once with SEC150, once with SEC250 (25 mM HEPES
pH 7.5, 250 mM NaCl, 1 mM DTT), once with SEC350 (25 mM HEPES pH 7.5, 350 mM NaCl,
1 mM DTT) and then twice with SEC150.
5m
Resuspend in 3 µL SEC150.
Add PI3KC3-C1, cofactors, and mCherry-2xFYVE as above (PI3KC3-C1 activity assay).
ATG8 lipidation assay
2h 35m
Use 0.5 µL GFP-Trap beads coated with ATG9A vesicles per 20 µL reaction.
Add 250 nanomolar (nM) each of: mCherry-GABARAPΔL117 (lipidatable), ATG7 (E1), ATG3 (E2), ATG5-ATG12-ATG16L1 (E3).
Add 2 millimolar (mM) ATP and 1 millimolar (mM) MgCl₂.
Incubate for 02:30:00 at 30 °C .
2h 30m
Wash beads once with SEC150.
Controls: non-lipidatable GABARAP-FL, mCherry alone, or omission of E1/E2/E3.
GST pull-down assay (ATG2A and ATG8)
1h
Equilibrate 5 µL Glutathione Sepharose beads with water and SEC150.
Incubate with 5 micromolar (µM) GST-tagged bait in SEC150 for 01:00:00 at 4 °C .
1h
Wash beads 3x with SEC150.
Resuspend in 5 µL SEC150.
Dispense 20 µL of 0.5 micromolar (µM) ATG2A-mCherry or ATG2A-LIR-mCherry into wells.
Add 1 µL beads per well.
Imaging: All samples were imaged using a Zeiss LSM 700 confocal microscopeequipped with a Plan Apochromat 20×/0.8 WD 0.55 mm objective.