Sep 08, 2025

Public workspaceTethering and lipid transfer assay

DOI
https://dx.doi.org/10.17504/protocols.io.eq2ly4nkplx9/v1
  • Elisabeth Holzer1
  • 1Laboratory of Sascha Martens, Max Perutz Labs, University of Vienna, Austria
Elisabeth Holzer
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DOI: https://dx.doi.org/10.17504/protocols.io.eq2ly4nkplx9/v1
Protocol Citation: Elisabeth Holzer 2025. Tethering and lipid transfer assay. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4nkplx9/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: August 01, 2025
Last Modified: September 08, 2025
Protocol Integer ID: 223995
Keywords: ASAPCRN, process of liposome tethering, liposome tethering, lipid transfer, tethering
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Abstract
This protocol describes the process of liposome tethering and lipid transfer.
Materials
Reaction Mix
AB
Donor liposomes (of 0.5 mg/mL total lipids)2.5 µL
Acceptor liposomes (of 0.5 mg/mL total lipids)2.5 µL
Proteins (final concentrations as per figure legends):
ATG2A250 nM
WIPI4100 nM
LC3B100 nM
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

Troubleshooting
Plate Setup and Reaction Conditions
Perform reactions in glass-bottom 384-well microplates (Greiner Bio-One, Cat# 781892).
Carry out all assays at TemperatureRoom temperature .
Temperature
Reaction Mix
Each Amount50 µL reaction (in SEC300 buffer) includes:
AB
Donor liposomes (of 0.5 mg/mL total lipids)2.5 µL
Acceptor liposomes (of 0.5 mg/mL total lipids)2.5 µL
Proteins (final concentrations as per figure legends):
ATG2A250 nM
WIPI4100 nM
LC3B100 nM


Mix
NBD Fluorescence Measurement (Lipid Transfer)
2h 20m 20s
Use a Tecan SPARK Multimode Microplate Reader (RRID:SCR_021897).
Record NBD fluorescence:

  • Excitation: 485 nm
  • Emission: 535 nm
  • Measurement interval: every Duration00:00:20
  • Duration: Duration01:30:00
1h 30m 20s
Add 0.5% (v/v) DDM to each well to fully solubilize lipids.

  • Amount2.5 µL of 10% (v/v) DDM to Amount50 µL reactions.
Pipetting
Measure maximum NBD fluorescence Duration00:50:00 after DDM addition.
50m
Normalize fluorescence:
  • Subtract the initial fluorescence value.
  • Divide by the maximal DDM-treated fluorescence.
Liposome Tethering (Optional Parallel Readout)
Measure absorbance at 405 nm every Duration00:00:20 in the same wells.
Analyze
This quantifies vesicle clustering (tethering) under identical conditions using the same plate reader.