Dynamic distribution of ASIC1a channels and other proteins within cells detected through fractionation

LIBIA CATALINA SALINAS CASTELLANOS; RODOLFO GABRIEL GATTO; SILVIA A MENCHÓN; MATÍAS BLAUSTEIN; OSVALDO D. UCHITEL; CARINA WEISSMANN

Membranes

Multidisciplinary Digital Publishing Institute

Año: 2022

2077-0375

Proteins in eukaryotic cells reside in different cell compartments. Many studies require the specific localization of proteins, as well as the detection of the dynamic changes in intracellular protein distribution. There are several methods available for this purpose that rely on the fractionation of the different cell compartments. Fractionation protocols have evolved ever since the use of a centrifuge to isolate organelles. In this study, we describe a simple method that involves the use of a tabletop centrifuge and different detergents to obtain cell fractions enriched in Cytosolic (Cyt), Plasma Membrane (PM), Membranous Organelles (MO), and nuclear (Nu) proteins and the identification of the proteins in each. Particularly, this method, serves to identify transmembrane proteins such as channel subunits as well as PM embedded or weakly associated proteins. This protocol uses a minute amount of cell material, typical equipment present at laboratories, and takes about 3 hours. The process was validated using endogenous and exogenously proteins expressed in the HEK293t cell line, and targeted to each compartment. We also show and quantify the shuttling of a protein channel from the MO to a PM fraction, and the shuttling of a kinase from a cytosolic to a nuclear location triggered via a specific stimulus.