Tissue-culture cell fractionation. Fractionation of membranes from tissue-culture cells homogenized by glycerol-induced lysis.
1. The disruption of various types of tissue-culture cells by (a) incubation in solutions of 1.2 M-glycerol and (b) transfer of the glycerol-loaded cells to relatively hypo-osmotic solutions of 0.25 M-sucrose was studied. 2. Bivalent cations (2mM-Mg2+) were generally included to preserve the nuclei, but some cells (polyoma-virus-transformed baby-hamster kidney cells) failed to be disrupted adequately under these conditions. 3. Other cells (mouse-embryo fibroblasts) required additional gentle Dounce homogenization to effect complete cell breakage. 4. Purification of the whole homogenate was carried out by a combination of differential centrifugation and sedimentation or flotation through sucrose gradients. 5. Enzyme analysis showed that plasma-membrane, endoplasmic-reticulum and mitochondrial fractions were obtained in good yield and purity.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D005347:Fibroblasts; D005990:Glycerol; D007668:Kidney; D051379:Mice; D013347:Subcellular Fractions / Q000201:enzymology
D005990:Glycerol