<ol><li>Giant unilamellar vesicle (GUV) Binding Assay<ol style="list-style-type:decimal;"><li>Using a cut tip, mix 5 &#xB5;l of in vitro translation reactions containing translated proteins and 5 &#xB5;l of GUVs in a tube and incubate at room temperature for 2-3 min.</li><li>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.</li><li>Place the 10 &#xB5;l mixture from step 1.1 into the small hole.</li><li>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.</li></ol></li></ol>

<table class="ck-table-resized"><tbody><tr><td>PDMS</td><td>Sylgard elastomer base kit, Dow-Corning</td><td>Sylgard, 184</td><td>&#xA0;</td></tr></tbody></table>

visualizing viral structural protein assembly on giant unilamellar vesicle membranes

Visualizing Viral Structural Protein Assembly on Giant Unilamellar Vesicle Membranes

Source: <a href="https://app.jove.com/v/54293/visualization-hiv-1-gag-binding-to-giant-unilamellar-vesicle-guv">Olety, B., et. al., Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes. J. Vis. Exp. (2016)</a>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.

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Begin with a translation reaction mixture containing YFP-tagged viral structural proteins.The viral structural protein is tagged with YFP for visualization and a fatty acid that enables membrane binding.Next, add an aliquot of the reaction mixture to a tube containing giant unilamellar vesicles (GUVs), large lipid spheres that mimic the cell membrane, and incubate.The fatty acid on the viral structural proteins interacts with the GUV lipid layer and allows attachment to its surface, forming a protein-GUV complex.Once bound, the viral structural proteins self-assemble on the vesicle membrane, mimicking the early steps of viral particle formation.Transfer the protein-GUV mixture into an imaging chamber and seal it with a coverslip to prevent the reaction mixture from evaporating.Using a fluorescence microscope, observe the protein-GUV complexes.The yellow-green signal at the boundary of the GUVs represents the assembly of viral structural proteins on their membrane.Once cooled, carefully bring the slide assembly to the bench and gently disassemble the chamber by removing the top slide with forceps. Transfer the GUVs with a clipped 1000 microliter pipette tip to a new tube. Handle this tube gently and use the GUVs within 90 minutes.Next, prepare an imaging chamber. Attach a PDMS sheet containing a small hole onto a clean cover slip. Press it down gently and firmly for a tight seal.Then, mix 5 microliters of the in vitro translation reaction with 5 microliters of GUVs. After a few minutes, transfer the mixture to the imaging chamber and proceed with imaging. To prevent evaporation, the imaging chamber can be covered with a cover slip.

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69 Views. Begin with a translation reaction mixture containing YFP-tagged viral structural proteins.The viral structural protein is tagged with YFP for visualization and a fatty acid that enables membrane binding.Next, add an aliquot of the reaction mixture to a tube containing giant unilamellar vesicles (GUVs), large lipid spheres that mimic the cell membrane, and incubate.The fatty acid on the viral structural proteins interacts with the GUV lipid layer and allows attachment to i...

Visualizing Viral Structural Protein Assembly on Giant Unilamellar Vesicle Membranes

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