title: Miniaturized, microarray-based assays for chemical proteomic studies of protein function.
abstract: Systematic analysis of protein and enzyme function typically requires scale-up of protein expression and purification prior to assay development; this can often be limiting. Miniaturization of assays provides an alternative approach, but simple, generic methods are in short supply. Here we show how custom microarrays can be adapted to this purpose. We discuss the different routes to array fabrication and describe in detail one facile approach in which the purification and immobilization procedures are combined into a single step, significantly simplifying the array fabrication process. We illustrate this approach by reference to the creation of arrays of human protein kinases and of human cytochrome P450s. We discuss methods for both ligand-binding and turnover-based assays, as well as data analysis on such arrays.
authors.affiliation: Division of Medical Biochemistry, Institute for Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa. jonathan.blackburn@uct.ac.za
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Blackburn
authors.forename: Aubrey
authors.initials: A
authors.lastname: Shoko
authors.forename: Natasha
authors.initials: N
authors.lastname: Beeton-Kempen
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006801:Humans; D055767:Immobilized Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055616:Microtechnology / Q000379:methods*; D019950:Mitogen-Activated Protein Kinase 1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000378:metabolism / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods*; D051578:Proto-Oncogene Proteins c-fes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D055767:Immobilized Proteins; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D019809:Streptavidin; D003577:Cytochrome P-450 Enzyme System; D051578:Proto-Oncogene Proteins c-fes; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1