RESUMO
Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labeled proteins can then be immunoprecipitated and analyzed by electrophoresis and gel imaging techniques. This chapter presents a protocol for the biosynthetic labeling and immunoprecipitation of pancreatic islet proteins which are known to be affected in disorders such as diabetes, obesity, and metabolic syndrome.
Assuntos
Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Marcação por Isótopo , Animais , Biomarcadores , Eletroforese , Imunoprecipitação , Técnicas de Imunoadsorção , Insulina/metabolismo , Marcação por Isótopo/métodos , Ratos , Radioisótopos de Enxofre/metabolismoRESUMO
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 with 35S-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.