{
  "abstract": "Small GTPases and many other peripheral membrane proteins regulate essential cellular processes through dynamic interactions with cellular membranes. These interactions are often controlled by membrane composition, particularly phosphoinositides (PIPs), which modulate the recruitment of GTPases, their regulators, and their effectors. Quantitative characterization of protein recruitment to defined membrane environments remains technically challenging. Here, we describe a protocol for preparing supported lipid bilayers (SLBs) containing defined phosphoinositide compositions in polydimethylsiloxane (PDMS) chambers and monitoring protein recruitment by total internal reflection fluorescence (TIRF) microscopy. Purified fluorescently labeled proteins are incubated with SLBs to measure membrane association in real time under controlled biochemical conditions. The method enables quantitative analysis of membrane recruitment kinetics and comparison of protein binding across different lipid compositions or in the presence of partner proteins. Combining the biochemical precision of a reconstituted membrane system with the sensitivity of TIRF imaging, this protocol provides a robust and versatile platform for studying lipid-dependent membrane recruitment. Although developed to investigate small GTPase signaling, it is readily applicable to a wide range of peripheral membrane proteins and membrane-associated signaling mechanisms. Key features • Reconstitutes small GTPase membrane recruitment on PIP-containing supported lipid bilayers compatible with TIRF microscopy. • Enables quantitative comparison of lipid-dependent binding across defined membrane compositions, including phosphoinositide species, phosphatidylserine content, and additional membrane components. • Suitable for purified small GTPases, GEFs, GAPs, and effectors, allowing direct testing of multicomponent membrane recruitment under controlled biochemical conditions. • Can be adapted for steady-state measurements, recruitment kinetics, and protein competition assays to dissect any membrane-dependent signaling mechanism.",
  "abstract_shown": true,
  "affiliations": [
    "CNRS, UMR8113, LBPA, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, Membrane Interfaces and Associated Diseases team, Gif-sur-Yvette, France.",
    "CNRS, UMR8531, PPSM, École Normale Supérieure Paris-Saclay, and Université Paris-Saclay, Gif-sur-Yvette, France.",
    "CNRS, UMR8113, LBPA, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, Bacterial Systems Biology and Anti-Microbial Resistance team, Gif-sur-Yvette, France."
  ],
  "article_type": "methods article",
  "arxiv": null,
  "author_total": 6,
  "authors": [
    {
      "affs": [
        0
      ],
      "name": "Cyril Pagès"
    },
    {
      "affs": [
        1
      ],
      "name": "Rasta Ghasemi"
    },
    {
      "affs": [
        0
      ],
      "name": "Marwan Zuber"
    },
    {
      "affs": [
        2
      ],
      "name": "Tatiana Coelho"
    },
    {
      "affs": [
        0
      ],
      "name": "Gérald Peyroche"
    },
    {
      "affs": [
        0
      ],
      "name": "Agata Nawrotek"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/5qxt2rgfag9tf.bib",
  "cited_by": 0,
  "doi": "10.21769/bioprotoc.5853",
  "fulltext_url": "https://europepmc.org/articles/PMC13710122",
  "id": "5qxt2rgfag9tf",
  "issns": [
    "2331-8325"
  ],
  "landing_url": "https://europepmc.org/article/MED/42947946",
  "language": "eng",
  "licence": "CC-BY-NC",
  "licence_label": "CC BY-NC",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by-nc/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "heading",
      "Biology"
    ],
    [
      "heading",
      "Clinical Protocols"
    ],
    [
      "heading",
      "Methods Article"
    ],
    [
      "openalex",
      "1312|Lipid Membrane Structure and Behavior"
    ]
  ],
  "openalex": "W7221110270",
  "pdf_url": "https://europepmc.org/articles/PMC13710122?pdf=render",
  "pmcid": "PMC13710122",
  "pmid": "42947946",
  "published": "2026-10-20",
  "publisher": "Bio-protocol, LLC",
  "ris": "https://research.draconitic.com/paper/5qxt2rgfag9tf.ris",
  "sources": [
    {
      "first_seen": "2026-10-31T00:20:34Z",
      "native_id": "MED:42947946",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42947946"
    },
    {
      "first_seen": "2026-10-31T12:21:09Z",
      "native_id": "PMC13710122",
      "source": "pmc",
      "url": "https://doi.org/10.21769/bioprotoc.5853"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1312|Lipid Membrane Structure and Behavior",
      "subfield": "molecular-biology"
    }
  ],
  "title": "Preparation of Phosphoinositide-Containing Supported Lipid Bilayers and Quantitative Assessment of Protein Recruitment to Membrane Surfaces.",
  "title_text": "Preparation of Phosphoinositide-Containing Supported Lipid Bilayers and Quantitative Assessment of Protein Recruitment to Membrane Surfaces.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-31T12:21:09Z",
  "url": "https://research.draconitic.com/paper/5qxt2rgfag9tf",
  "venue": "Bio-protocol",
  "versions": []
}