title: Capture and qualitative analysis of the activated Fc receptor complex from live cells.
abstract: This unit describes the isolation of activated Fc receptor complexes from RAW 264.7 macrophages using live-cell affinity receptor chromatography (LARC). The Fc receptor complex is activated and captured by IgG-coated microbeads on the surface of live macrophages. After the cells are disrupted, the receptor complexes are isolated by washing and sucrose gradient ultracentrifugation. Soluble proteins associated with the receptor complex are then eluted from the beads using a stepwise series of salt buffers and aqueous acetonitrile. The eluted proteins and the residual insoluble proteins on the beads can then be digested with trypsin and subjected to liquid chromatography, electrospray ionization, and tandem mass spectrometry (LC-ESI-MS/MS). Controls include IgG-coated beads incubated with crude cell lysates or growth medium and beads coated with oxidized LDL or bovine serum albumin. Using this method, proteins present in IgG-FcR complexes can be distinguished from those in control scavenger receptor complexes (oxLDL or BSA). Thus, LARC is capable of detecting specific members of IgG receptor supramolecular complexes.
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, Ontario, Canada.
authors.forename: Angelica K
authors.initials: AK
authors.lastname: Florentinus
authors.forename: Peter
authors.initials: P
authors.lastname: Bowden
authors.forename: Veronica
authors.initials: V
authors.lastname: Barbisan
authors.forename: John
authors.initials: J
authors.lastname: Marshall
mesh_terms: D000818:Animals; D000936:Antigen-Antibody Complex / Q000737:chemistry*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D003285:Contractile Proteins / Q000737:chemistry; D064448:Filamins; D007074:Immunoglobulin G / Q000737:chemistry; D008024:Ligands; D008264:Macrophages / Q000737:chemistry*; D051379:Mice; D008840:Microfilament Proteins / Q000737:chemistry; D008863:Microspheres; D046912:Multiprotein Complexes / Q000032:analysis / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011961:Receptors, Fc / Q000032:analysis* / Q000737:chemistry; D017452:Receptors, IgG / Q000737:chemistry; D051116:Receptors, Scavenger / Q000737:chemistry; D012995:Solubility; D053719:Tandem Mass Spectrometry; D014461:Ultracentrifugation / Q000379:methods
chemical_list: D000936:Antigen-Antibody Complex; D003285:Contractile Proteins; D064448:Filamins; D007074:Immunoglobulin G; D008024:Ligands; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D011961:Receptors, Fc; D017452:Receptors, IgG; D051116:Receptors, Scavenger