Mar 03, 2026
Liposome Binding Assay with GFP-CHMP4B
- Andrea Paquola1,2,
- Clare E. Benson1,
- Smita Eknath Desale1,
- Cagakan Ozbalci1,
- Elisabeth M. Storck1,
- Stephen J. Terry1,
- Bhagyashree Dasari Rao1,
- Kelechi Nwite1,
- Federica Ferrentino1,
- Ulrike S. Eggert1,2
- 1Randall Centre for Cell and Molecular Biophysics, King’s College London;
- 2Department of Chemistry, King’s College London
Protocol Citation: Andrea Paquola, Clare E. Benson, Smita Eknath Desale, Cagakan Ozbalci, Elisabeth M. Storck, Stephen J. Terry, Bhagyashree Dasari Rao, Kelechi Nwite, Federica Ferrentino, Ulrike S. Eggert 2026. Liposome Binding Assay with GFP-CHMP4B. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8xk39v2w/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: February 18, 2026
Last Modified: March 03, 2026
Protocol Integer ID: 243628
Keywords: liposome binding assay with gfp, generation of giant unilamellar vesicle, liposome binding assay, giant unilamellar vesicle, membrane remodelling, membrane, tagged protein, percentage of membrane, specific phosphoinositide composition, based immunoaffinity purification, immunoaffinity purification, hela cell lysate, protein
Funders Acknowledgements:
Wellcome Trust Investigator Award
Grant ID: 110060/Z/15/Z
BBSRC grants
Grant ID: BB/X000605/1 and BB/V003518/1
Abstract
This protocol provides an organized
workflow for reconstituting and quantifying ESCRT-III (GFP-CHMP4B) membrane
binding. We detail the generation of Giant Unilamellar Vesicles (GUVs) with
specific phosphoinositide compositions including PI(3)P and PI(4)P through
electroformation. The procedure includes an enrichment of GFP-tagged proteins
from HeLa cell lysates using magnetic bead-based immunoaffinity purification,
followed by an acidic elution and immediate neutralization to preserve protein
stability. Interaction is validated through a Liposome Binding Assay (LBA)
using ultracentrifugation and densitometric Western blot analysis to determine
the percentage of membrane-associated protein. Adhering to GPP3 and ICMJE
standards, the systematic approach ensures high reproducibility and data
integrity for biophysical research in membrane remodelling and cargo sorting.
Guidelines
Technical Note: The density difference between the internal sucrose and external glucose allows the GUVs to
sink to the bottom.
Troubleshooting
Part 1: Preparation of Giant Unilamellar Vesicles (GUVs) via Electroformation
Prepare the lipid mixture (total: 200 nmol) in the defined ratios: Composition A: 100% POPC, Composition B: POPC / PI(3)P (90:10 mol/mol) or Composition C: POPC / PI(4)P (90:10 mol/mol). All lipids were obtained from Avanti Polar Lipids.
Spread the lipid mixture onto the conductive side of an ITO-coated glass slide.
Place the slide in a vacuum chamber for ~2.5 hours to ensure complete evaporation of the organic solvent.
Apply a thin layer of vacuum grease to a medium O-ring (18 mm inner diameter) and place the O-ring around the dried lipid film on the slide.
Gently add 270 µl of 250 mM sucrose (Fluorochem) into the O-ring area.
Place a second ITO-coated slide (conductive side down) on top of the O-ring to create a "sandwich" assembly.
Insert the assembly into the Nanion Vesicle Prep Pro (Nanion Technologies) and run the standard 120-minute protocol with the following settings: Temperature: 55°C, Voltage: 3 V, Frequency: 10 Hz, Rise/Fall Time: 3 minutes
Carefully collect the GUV suspension and store at 4°C. For best results, visualize within 24 hours.
Prepare an ibidi 8-well microscope chamber (ibidi, Gräfelfing, Germany) by filling it with ~400 µl of 250 mM glucose (Sigma-Aldrich).
Slowly suspend 20 µl of the GUV solution into the glucose-filled chamber.
Allow the vesicles to settle at the bottom of the chamber for 2 hours before starting imaging.
Part 2: Enrichment of GFP-CHMP4B from HeLa Cells
Seed HeLa cells stably expressing GFP-CHMP4B into 15-cm Petri dishes at a density of 20-30 x 106 cells per dish.
Detach cells in 1ml KPBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4) using a cell scraper, and transfer the suspension (1ml) to a chilled tube, keep on ice.
Sonicate using probe sonicator. Sonication parameters: 20 s ON / 30 s OFF, total of 3 min, at 30 kHz, keeping samples on ice.
Clarify the cell lysate by centrifugation at 3,500×g for 10 min at 4 °C to remove debris and collect the resulting supernatant.
Add 35 µl of GFP magnetic beads (Chromotek) to the entire clarified supernatant (~1 ml) after centrifugation and incubate for 1 hour at 4 °C with gentle rotation.
Place the tubes on a magnetic stand to collect the beads and discard the supernatant containing unbound proteins.
Wash the beads twice with 500 μL ice-cold KPBS to ensure all non-specific proteins are removed.
Perform a brief wash of the beads using ice-cold PIPES wash buffer (20mM, pH=6.8) for 1–2 minutes at 4 °C.
Add 100 µl of 0.1 M Glycine-HCl (pH 2.5) to the beads and incubate for 1–2 minutes at 4 °C with gentle rotation for acidic extraction.
Rapidly collect the supernatant and immediately neutralize it by adding 10 µl (1/10 volume) of 1 M Tris-HCl (pH 8.5) to preserve protein stability.
Part 3: Liposome Binding Assay (LBA)
Prepare POPC (100%), POPC/PI4P (90/10), or POPC/PI3P (90/10) GUVs using the electroformation method described in Part 1.
Combine 65 nmol (approx. 50 µg) of the prepared GUVs with 20 µg of the neutralized, eluted GFP-CHMP4B protein in a 1.5ml Eppendorf tube.
Incubate the protein-lipid mixture at room temperature for 30 minutes.
Centrifuge the mixture at 100,000 x g for 30 minutes at 20 °C to separate liposome-bound protein (pellet) from unbound protein (supernatant).
Carefully fractionate the supernatant and the pellet for downstream analysis.
Process a "Total Protein" control sample in parallel following the same steps but in the absence of liposomes.
Subject the supernatant, pellet, and total protein fractions to Western blotting, probing with a GFP antibody for CHMP4B detection.
Quantify the intensities of the resulting bands using ImageJ software.
Calculate the percentage of liposome-bound protein by comparing the pellet fraction intensity to the total protein control intensity.
Protocol references
Rao, B. D. et al. Horizontal acquisition of prokaryotic hopanoid biosynthesis reorganizes membrane physiology driving lifestyle innovation in a eukaryote. Nat. Commun. 16, 3291 (2025). DOI: 10.1038/s41467-025-58515-w
Boyd, M. A. & Kamat, N. P. Visualizing tension and growth in model membranes using optical dyes. Biophys. J. 115, 1307-1315 (2018). DOI: 10.1016/j.bpj.2018.08.021
Efimova, S. S. et al. Regulation of the pore-forming activity of cecropin a by local anesthetics. Cell Tissue Biol. 12,
331-341 (2018). DOI: 10.1134/S1990519X18040028
Wollert, T. & Hurley, J. H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.
Nature 464, 864-869 (2010). DOI: 10.1038/nature08391