Cytoplasmic extract homogenization

Despite the elaborate washing procedures usually employed, cell-wall preparations may also frequently be contaminated with cytoplasmic constituents that sediment with the walls after tissue homogenization. Starch grains and proteins are particularly difficult to remove in this respect. Both incubation with purified alpha amylase (EC 3.2.1.1)55,101 and extraction with chloral hydrate118 have been utilized for removal of starch from cell-wall preparations. Pronase57,101 has been used to remove proteins. 

Root tip homogenization, cytoplasmic protein extraction, gel electrophoresis, and densitometry analysis... 

Lactate dehydrogenase is found in the cytoplasm and can be liberated into solution when the cells are broken with a homogenizer or by osmotic shock. The procedure introduced by Straub (5) consisted of extraction of the enzyme, adsorbing onto calcium phosphate gel, and fractionating in a series of acetone and ammonium sulfate steps. The method of Jecsai 106) gives crystalline M4 enzyme from pig skeletal muscle simply by repeated ammonium sulfate fractionation. Although much refinement was achieved with these methods, and multienzyme isolation procedures were... 

The key step in LC isolation is the preparation of pure smooth muscle myosin (Hasegawa et al., 1988). This is usually executed by homogenizing the muscle mince with salt solutions (60-100 mM ionic strength) to remove the cytoplasmic proteins and to a certain extent the thin filament proteins. A crude actomyosin is then extracted form the well-washed muscle residue with salt solutions containing ATP and the actin component is removed by ultracentrifugation. The LCs are... 

Egg cytoplasmic MVs forming the bulk of the sea urchin male pronudear envelope in vivo are derived from the endoplasmic reticulum (ER) (Congo and Anderson, 1968). Egg homogenate MVs, also largely derived from the ER, are required for nuclear envelope assembly in vitro (Collas and Poccia, 1995a, 1996b). MVs can be easily isolated from the cytoplasmic S o extract. 

Preparation of oocyte samples and immunoprecipitation. Extracts containing P-labeled RNAs are prepared either from whole oocytes or isolated nuclear and cytoplasmic fractions by homogenization on ice in a suitable buffer (e.g., 40 mM HEPES, pH 7.9, with KOH 100 mM potassium acetate, 8% (v/v) glycerol, 5 mM MgS04, 2 mM EGTA, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5% (v/v) aprotinin, 2 /ig/ml of each leupeptin and pepstatin, and 0.1 U/jul of RNasin) at a ratio of at least 25 pi of buffer per oocyte (or isolated cytoplasm) or as little as 1-4 pi per isolated nucleus. If the antibody recognizes RNA rather than an... 

Sequential extraction of cells to recover lysates enriched in cytosolic and nuclear proteins 268 Controlled cell lysis to produce a post-nuclear supernatant homogenate of cytoplasmic proteins 269... 

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