title: Cell surface biotinylation of receptor tyrosine kinases to investigate intracellular trafficking.
abstract: Cell surface biotinylation is a biochemical approach to covalently bind membrane-impermeable biotin to the extracellular domain of membrane proteins, such as receptor tyrosine kinases (RTKs). Subsequent to ligand incubation periods, activated biotinylated receptors may internalize from the cell surface into early endosomes and then travel through intracellular compartments to either recycle back to the membrane or degrade in lysosomes. The biotin-labeled proteins may be detected through affinity purification with streptavidin agarose resins. This chapter describes methods for cell surface biotinylation to assess RTK trafficking steps.
authors.affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Botterell Hall Rm A315, Kingston, ON, Canada, K7L 3N6.
authors.forename: Mathieu J F
authors.initials: MJ
authors.lastname: Crupi
authors.forename: Douglas S
authors.initials: DS
authors.lastname: Richardson
authors.forename: Lois M
authors.initials: LM
authors.lastname: Mulligan
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D021381:Protein Transport; D020794:Receptor Protein-Tyrosine Kinases / Q000737:chemistry* / Q000378:metabolism; D012685:Sepharose / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry; D013388:Succinimides / Q000737:chemistry*
chemical_list: D013388:Succinimides; C094773:sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; D001710:Biotin; D012685:Sepharose; D019809:Streptavidin; D020794:Receptor Protein-Tyrosine Kinases