title: Two Dimensional Gel Electrophoresis of Insulin Secretory Granule Proteins from Biosynthetically-Labeled Pancreatic Islets.
abstract: Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with35S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE. This provides a means of determining whether or not the biosynthetic rates of the entire granule constituents are coordinately regulated.
authors.affiliation: Laboratory of Neuroproteomics, Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas (UNICAMP), Rua Monteiro Lobato 255 F/01, Cidade Universitária Zeferino Vaz, 13083-862, Campinas, Brazil.
authors.forename: Paul C
authors.initials: PC
authors.lastname: Guest
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism*; D013347:Subcellular Fractions; D013462:Sulfur Radioisotopes
chemical_list: D007328:Insulin; D013462:Sulfur Radioisotopes
keywords: 2D electrophoresis; Insulin secretory granules; Pancreatic islets; Pulse-chase radiolabeling; Subcellular fractionation