Supported planar bilayers for study of the immunological synapse.
Supported planar bilayers have been used in immunology research for over 25 years, including in the initial demonstrations of MHC-peptide complex functional activity and adhesion molecule activity. More recent modifications of the method have been used to measure two-dimensional affinities and to study the formation of the immunological synapse. This unit covers the incorporation of glycolipid-anchored membrane proteins, 6-histidine-tagged soluble proteins, and monobiotinylated soluble proteins into supported planar bilayers. Reagents developed for the MHC-peptide tetramer staining method (UNIT 17.3) can readily be adapted to presentation on planar bilayers. The unique advantage of this approach is that the proteins presented on the surface of the supported bilayer are laterally mobile. This provides a more physiological presentation of cell-surface molecules and supports visualization of protein rearrangement on the bilayer by live cells.
Dustin
Michael L
ML
Skirball Institute of Biomolecular Medicine, New York, New York.
Starr
Toby
T
Skirball Institute of Biomolecular Medicine, New York, New York.
Varma
Rajat
R
Skirball Institute of Biomolecular Medicine, New York, New York.
Thomas
V Kaye
VK
Skirball Institute of Biomolecular Medicine, New York, New York.
D000818:Animals; D017951:Antigen Presentation; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000302:isolation & purification; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000276:immunology*; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D011948:Receptors, Antigen, T-Cell / Q000276:immunology* / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology*
D005456:Fluorescent Dyes; D017261:Glycosylphosphatidylinositols; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell