Sep 08, 2025

Tethering 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

Plate Setup and Reaction Conditions
Perform reactions in glass-bottom 384-well microplates (Greiner Bio-One, Cat# 781892).
Carry out all assays at Room temperature .
Reaction Mix
Each 50 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


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 00:00:20
  • Duration: 01:30:00
1h 30m 20s
Add 0.5% (v/v) DDM to each well to fully solubilize lipids.

  • 2.5 µL of 10% (v/v) DDM to 50 µL reactions.
Measure maximum NBD fluorescence 00:50:00 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 00:00:20 in the same wells.
This quantifies vesicle clustering (tethering) under identical conditions using the same plate reader.