Subcellular fractionation of suspension Chinese Hamster Ovary (CHO) cells v1

Subcellular fractionation is a widely adopted technique to study cellular biology, and localization and functions of several proteins , and, more recently, it has been applied previous to proteomics to quantify low concentration proteins, and characterize many subcellular organelles . Since CHO cells play a central role for recombinant protein (RP) production, a deeper study of their cellular biology is mandatory in order to construct new cell sub-lines with a more robust RP expression phenotype. Therefore, the goal of the present protocol is to provide the necessary basis for separation of subcellular organelles from suspension Chinese hamster ovary (CHO) cells for a subsequent proteomic study of isolated fractions. Differential and isopycnic centrifugation based sections are presented for enrichment of different subcellular compartments, together with a precipitation section for recovering of proteins from isolated fractions. Isopycnic centrifugation is based on discontinuos sucrose gradients, of which one is adapted from the literature for the separation of nuclear and mitochondrial precipitates , and the other is designed in our laboratory for the separation of the microsomal precipitate. This protocol provides a novel discontinuous sucrose gradient for the separation of the components of the microsomes, which increases the separation efficiency with respect to previously reported protocols .