title: Single-molecule optical methods analyzing receptor tyrosine kinase activation in living cells.
abstract: Receptor tyrosine kinase activity is typically measured by diverse biochemical methods detecting the amount of phosphorylation of proteins within a cell lysate. In this chapter, we present biophysical methods that allow for studying the activation process of single receptors, in particular the human epidermal growth factor receptor (EGFR) family, in live cells. We describe optical tracking of quantum dot (QD)-labeled single receptors using the total internal reflection fluorescence microscopy (TIRFM), and initial steps of data analysis to identify the time-dependent variation of single-receptor diffusion, which can be widely applied to studying activation of various cell surface receptors.
authors.affiliation: Research Oncology, Genentech, Inc., 1 DNA Way, Mail Stop 72A, South San Francisco, CA, 94080, USA, inheec@gene.com.
authors.forename: Inhee
authors.initials: I
authors.lastname: Chung
authors.forename: Ira
authors.initials: I
authors.lastname: Mellman
mesh_terms: D045744:Cell Line, Tumor; D004058:Diffusion; D004789:Enzyme Activation / Q000187:drug effects; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D066246:ErbB Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D009038:Motion; D017434:Protein Structure, Tertiary; D045663:Quantum Dots / Q000737:chemistry; D013816:Thermodynamics; D059008:Time-Lapse Imaging
chemical_list: D018796:Immunoconjugates; D007140:Immunoglobulin Fab Fragments; D004815:Epidermal Growth Factor; C512478:EGFR protein, human; D066246:ErbB Receptors