Subcellular fractionation of zebrafish embryos and mitochondrial calcium uptake application

Abstract

The zebrafish model is a powerful tool for studying developmental and apoptotic processes in vertebrates. Recently we showed the critical role of two members of the Bcl-2 family of proteins during early zebrafish development. Bcl-2 proteins can be localized in different cellular compartments including mitochondria and endoplasmic reticulum and their functional characterization often requires specific subcellular membrane fractionation. Although many protocols describe in detail the subcellular fractionation from cells or tissues, none is specifically tuned to the fractionation from zebrafish embryos. Here we provide a protocol designed for mitochondria and endoplasmic reticulum membrane isolation from zebrafish embryos and subsequent application like mitochondrial calcium uptake. Fractionation is performed by differential centrifugation and distribution of proteins of interest in the corresponding fractions is subsequently analyzed. After the preparation of embryos extracts, the subcellular fractionation requires 4 h and assay for mitochondrial calcium uptake can be performed in 2 h from isolated mitochondria.