Liposome Binding Assay with GFP-CHMP4B v1

This protocol provides an organizedworkflow for reconstituting and quantifying ESCRT-III (GFP-CHMP4B) membranebinding. We detail the generation of Giant Unilamellar Vesicles (GUVs) withspecific phosphoinositide compositions including PI(3)P and PI(4)P throughelectroformation. The procedure includes an enrichment of GFP-tagged proteinsfrom HeLa cell lysates using magnetic bead-based immunoaffinity purification,followed by an acidic elution and immediate neutralization to preserve proteinstability. Interaction is validated through a Liposome Binding Assay (LBA)using ultracentrifugation and densitometric Western blot analysis to determinethe percentage of membrane-associated protein. Adhering to GPP3 and ICMJEstandards, the systematic approach ensures high reproducibility and dataintegrity for biophysical research in membrane remodelling and cargo sorting.