Topography of the total protein population from cultured cells upon fractionation by chemical extractions

Abstract

1. Chemical extractions are proposed a s a major tool for a fractionation of cellular proteins. As a model system, proteins from cultured hamster lens cells have been divided by independent extractions into seven subcellular fractions, corresponding to water-soluble proteins and the proteins from membranes, microfilaments (and, other deoxycholate-soluble proteins), intermediate filaments, microtubules, polysomes and nuclei respectively. The latter two fractions have becn subfractionated yielding ribosomal proteins, the elongation and initiation f, act o r s of the protein-synthesis machinery, chromatin proteins and non-chromatin proteins.2. The protein compositions of the fractions have been analyzed by one-dimensional and two-dimensional gel electrophoresis. This resulted in an almost complete topography of the proteins detected on two-dimensional gels of total-cell lysates.3. Comparison of two-dimensional patterns of proteins from the total-cell lysate and proteins from hamster erythrocytes or from liver, muscle or brain tissue showed that the different cell types have only few proteins in common. Two proteins are common to all of these cell types, namely actin and a 68-kDa protein. The latter protein was, like actin, vimentin and the tubulin subunits, also present in most cell fractions. Evidence is presented that this protein is identical to a 68-kDa heat-shock protein.

Selective Gene Expression in Focal Cerebral Ischemia

“…This similarity and the favorable proximity to limited arterial oxygen suggest that the vasculature is an important source of residual protein synthesis in regions of severe ischemia. This hypothesis is consistent with the tentative identification by 2D-PAG E criteria of P55 as vimentin, an intermediate filament synthesized by mesenchymal cells (Hightower and White, 1982;Lenstra and Bloemendal, 1983;Pearce et al, 1983).…”

“…Subtle regional changes in the cortical proteins, however, may not have been detected. Although most pro teins could not be identified, the location and rela tive concentrations of some proteins were suffi ciently characteristic to permit their identification based on reported mobilities in similar 2D-PAG E systems (Wilson et aI., 1977;Strocchi et aI., 1981Strocchi et aI., , 1982Gilbert and Strocchi, 1983;Heydorn et aI., 1983;Lenstra and Bloemendal, 1983;Pearce et aI., 1983;Perry et aI., 1983). Actin, tubulin, creatine Right Hemisphere kinase, and a major cellular protein, P73, are la beled (Fig.…”

Differential detergent fractionation of isolated hepatocytes: Biochemical, immunochemical and two‐dimensional gel electrophoresis characterization of cytoskeletal and noncytoskeletal compartments

“…Detergent fractionation involves the sequential extraction of tissues or cells with detergentcontaining buffers, and partitions cellular proteins into functionally distinct compartments [ 15,[19][20][21][22] while simultaneously preserving cytoskeletal integrity and cytoskeletal-noncytoskeletal interactions. Like differential pelleting, detergent fractionation is useful for confirming the subcellular distribution of proteins or metabolites, monitoring compartmental redistributions of target compounds and for enhancing the detectability of low-abundance species.…”

Expression and Solubilization of Insect Cell-Based Rabies Virus Glycoprotein and Assessment of Its Immunogenicity and Protective Efficacy in Mice

“…Since the expressed r-RVG is membrane bound, attempts were made to isolate the membrane protein, which is a tedious, time-consuming, and cumbersome process (14,16). Effective cell lysis and protein extraction from different cell and tissue types require the correct choice not only of detergents but also of buffers.…”