Frozen tissue can provide reproducible proteomic results of subcellular fractionation

Analytical subcellular fractionation of jejunal biopsy specimens: methodology and characterization of the organelles in normal tissue.

1. Portions of closed jejunal biopsies were gently homogenized in isotonic sucrose or sorbitol and centrifuged at 800 g for 10 min to prepare a cell extract. 2. The extract was fractionated in a single-step procedure by isopycnic centrifugation on a continuous sucrose or sorbitol density gradient with the Beaufay automatic zonal rotor. 3. The subcellular organelles were located in the density g...

Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells

Many types of studies require the localization of a protein to, or isolation of enriched protein from a specific cellular compartment. Many protocols in the literature and from commercially available kits claim to yield pure cellular fractions. However, in our hands, the former often do not work effectively and the latter may be prohibitively expensive if a large number of fractionations are re...

Implications for Subcellular Fractionation in Proteomics

Functional proteome analysis is not restricted to the sequence information but includes the broad spectrum of structural modifications and quantitative changes of proteins to which they are subjected in different tissues and cell organelles and during the development of an organism. Cell biology has provided the means required for the analysis of the composition and properties of purified cellu...

Principles of Tissue Fractionation

Analytical subcellular fractionation of needle-biopsy specimens from human liver.

1. Fragments (2-20 mg wet wt.) of closed needle-biopsy specimens from human liver were disrupted in iso-osmotic sucrose and subjected to low-speed centrifugation. The supernatant was layered on a linear sucrose-density gradient in the Beaufay small-volume automatic zonal rotor. The following organelles, with equilibrium densities (g/ml) and principal marker enzyme shown in parentheses, were res...