Visualizing Viral Structural Protein Assembly on Giant Unilamellar Vesicle Membranes

Résumé

Source: Olety, B., et. al., Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes. J. Vis. Exp. (2016)

This study demonstrates a fluorescence-based assay to observe the membrane-binding behavior of human immunodeficiency virus type-1 structural protein. Using synthetic giant unilamellar vesicles as model membranes, the assay visualizes protein recruitment and assembly, mimicking early steps of viral particle formation.

Protocole

1. Giant unilamellar vesicle (GUV) Binding Assay
   1. Using a cut tip, mix 5 µl of in vitro translation reactions containing translated proteins and 5 µl of GUVs in a tube and incubate at room temperature for 2-3 min.
   2. Attach a polydimethylsiloxane (PDMS) sheet with a small hole (3-4 mm diameter) on a clean cover slip and ensure a tight seal by gently and firmly pressing it against the coverslip.
   3. Place the 10 µl mixture from step 1.1 into the small hole.
   4. Image the mixture under an inverted epi- or confocal fluorescence microscope at room temperature.
      NOTE: To prevent evaporation, the imaging chamber can be covered by a coverslip.

Matériaux

Liste des matériaux utilisés dans cet article

Nom	Entreprise	Numéro de catalogue	Commentaires
PDMS	Sylgard elastomer base kit, Dow-Corning	Sylgard, 184