JoVE Encyclopedie van Experimenten
Biologische Technieken
0 weergaven • 3:05 min. • July 8th, 2025
Bacterial integral membrane proteins, anchored to lipid bilayer membranes, facilitate transport of molecules across membranes and transduction of signals. For structural studies, specific integral membrane recombinant proteins are overexpressed with fused green fluorescent protein, GFP.
To evaluate recombinant membrane protein stability following detergent solubilization, obtain bacterial membrane fraction containing GFP-tagged integral membrane proteins. Add a non-ionic detergent. Beyond the critical concentration, the detergent forms micelles.
Micelles interact with lipid membranes and membrane proteins, solubilizing the membranes and forming protein-detergent complexes. Additionally, membrane proteins' hydrophobic transmembrane regions may remain unmasked, causing protein aggregation. Further, free GFPs may result due to degradation of membrane proteins.
Centrifuge the mixture. Collect the detergent-solubilized fraction-containing supernatant.
Assemble a size-exclusion chromatography column connected to a fluorescence detector. The column matrix comprises agarose beads with pore sizes defining a broad fractionation range.
Equilibrate the column with detergent-containing buffer
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