Microplate-based analysis of protein-membrane binding interactions via immobilization of whole liposomes containing a biotinylated anchor.
Cellular membranes play key roles in the regulation of a range of important biological processes. However, the characterization of membrane involvement in these events is difficult to achieve due to the complexity of the membrane bilayer and the challenges associated with handling and analyzing these systems. As such, rapid and reliable approaches for characterizing membrane-based processes are necessary. To address this issue, we have first developed an azide-tagged modular lipid anchor scaffold (2) that can be conveniently derivatized via click chemistry to functionalize the membrane surface. This was used to access biotin- and fluorescein-lipid conjugates 1a and 1b, respectively. These compounds were then employed to perform and characterize the immobilization of liposomes containing biotin-anchor 1a onto streptavidin-coated microplates. Results from these studies indicated clean, biotin-dependent surface deposition. This strategy for liposome attachment was then applied to a microplate-based platform to detect the binding of receptor proteins to immobilized liposomes, specifically for the membrane binding of protein kinase Calpha (PKCalpha). The resulting data indicated direct detection of binding to the membrane-functionalized surface. The reported approaches provide efficient methods for the derivatization of the membrane surface, which is applicable to the study of membrane-based processes. Finally, the described microplate-based liposome binding assay allows for high-throughput analysis of important protein-membrane binding events.
Losey
Erin A
EA
Department of Chemistry, the University of Tennessee, Knoxville, Tennessee 37996, USA.
Smith
Matthew D
MD
Meng
Meng
M
Best
Michael D
MD
D000818:Animals; D019921:Biotinylation; D002462:Cell Membrane / Q000378:metabolism*; D006735:Horseradish Peroxidase / Q000378:metabolism; D007074:Immunoglobulin G / Q000276:immunology / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D011485:Protein Binding; D051571:Protein Kinase C-alpha / Q000276:immunology / Q000378:metabolism
D007074:Immunoglobulin G; D008081:Liposomes; D008565:Membrane Proteins; D006735:Horseradish Peroxidase; D051571:Protein Kinase C-alpha