In this study, we report different protocols used to obtain highly enriched and well-characterized protein fractions that could be used to determine the subcellular localization of proteins. Different protein fractions (total, cytosolic, total membrane, sarcolemmal, and nuclear) were isolated from mouse heart by a combination of either polytron homogenization or liquid nitrogen pulverization followed by density gradient centrifugation. Triton X-100 was used in specific fractions to help in the solubilization of proteins obtained with fractionation protocols. Following the isolation, enzymatic assays and Western blot analysis were used to evaluate the enrichment and/or cross-contamination of these protein fractions. Glucose-6-phosphate dehydrogenase, Na+/K+-ATPase, mitochondrial Ca2+-ATPase, sarco-endoplasmic reticulum Ca2+-ATPase, glucose-regulated protein, and nucleoporin P62 were used as specific markers for the cytosol, sarcolemma, mitochondria, sarco-endoplasmic reticulum, endoplasmic reticulum, and nucleus, respectively. The results show that we obtained enriched protein fractions with little to no cross-contamination. These purification protocols allow us to obtain different protein fractions that could be used in a wide variety of studies.