title: 2D Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically Labelled Pancreatic Islets.
abstract: Pulse radiolabelling of cells with radioactive amino acids such is a common method for investigating the biosynthetic rates of proteins. In this way, the abundance of newly synthesized proteins can be determined by several proteomic techniques including 2D gel electrophoresis (2DE). This chapter describes a protocol for labelling pancreatic islets with35S-methionine in the presence of low and high concentrations of glucose, followed by subcellular fractionation enrichment of secretory granule proteins and analysis of the granule protein contents by 2DE. This demonstrated that the biosynthetic rates of most of the granule proteins are co-ordinately regulated in the presence of stimulatory glucose concentrations.
authors.affiliation: Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Cidade Universitária Zeferino Vaz, Rua Monteiro Lobato 255 F/01, 13083-862, Campinas, Brazil. paulcguest@yahoo.com.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Guest
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002469:Cell Separation / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000032:analysis* / Q000096:biosynthesis; D007515:Islets of Langerhans / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D008715:Methionine / Q000032:analysis; D051381:Rats; D022142:Secretory Vesicles / Q000737:chemistry*; D013462:Sulfur Radioisotopes / Q000032:analysis
chemical_list: D007328:Insulin; D013462:Sulfur Radioisotopes; D008715:Methionine; D005947:Glucose
keywords: 2DE; Insulin secretory granules; Pancreatic islets; Proteomics; Pulse-chase biosynthetic labelling; Subcellular fractionation