RESUMEN
Pulse radiolabelling of cells with radioactive amino acids is a common method for studying the biosynthesis of proteins. The labelled proteins can then be immunoprecipitated and analysed by electrophoresis and imaging techniques. This chapter presents a protocol for the biosynthetic labelling and immunoprecipitation of pancreatic islet proteins which are known to be affected in psychiatric disorders such as schizophrenia.
Asunto(s)
Inmunoprecipitación/métodos , Insulina/análisis , Islotes Pancreáticos/química , Proproteína Convertasa 2/análisis , Vesículas Secretoras/química , Especificidad de Anticuerpos , Cromatografía en Agarosa/métodos , Electroforesis/métodos , Glucosa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Inmunoprecipitación/instrumentación , Inmunoadsorbentes , Insulina/biosíntesis , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Marcaje Isotópico/métodos , Metionina/análisis , Proproteína Convertasa 2/biosíntesis , Vesículas Secretoras/enzimología , Radioisótopos de Azufre/análisis , UreaRESUMEN
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 with 35S-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.