A procedure for the enrichment and isolation of Halobacterium

A procedure for the specific enrichment and isolation of species of the genus Halobacterium was designed, based on the ability of Halobacterium cells to grow anaerobically by fermentation of l-arginine. None of the other genera of neutrophilic halophilic Archaea tested grew fermentatively on arginine. Using anaerobic enrichments in the presence of arginine, representatives of the genus Halobacterium were consistently isolated from saltern crystallizer ponds in Eilat (Israel) and San Francisco Bay (California), environments in which Halobacterium represents only a very small fraction of the halophilic archaeal community.     

A rapid procedure for the isolation of phycobilisomes from cyanobacteria

This paper describes a method for the rapid isolation of phycobilisomes using a cationic detergent, CTAB (cetyltrimethylammonium bromide). The method has distinct advantages over those currently in use in that (i) release of intact phycobilisomes from cells in the presence of CTAB occurs in 40 s (as compared to 40-60 min of incubation required with Triton X-100), thereby reducing the chances of proteolysis of the component phycobiliproteins; and (ii) these phycobilisome preparations have reduced chlorophyll contamination in the initial stages. In addition this method also helps retain the structural and functional properties, as evidenced by spectroscopy and sodium dodecyl sulfate-polyacrylamide gel analysis.     

A simple procedure for the isolation of protein disulphide-isomerase.

A two-step procedure is described for the purification of protein disulphide-isomerase (PDI). This procedure is based on the previous finding that the beta-subunit of the prolyl 4-hydroxylase tetramer (alpha 2 beta 2) is identical with PDI [Koivu, Myllylä, Helaakoski, Pihlajaniemi, Tasanen & Kivirikko (1987) J. Biol. Chem. 262, 6447-6449; Pihlajaniemi, Helaakoski, Tasanen, Myllylä, Huhtala, Koivu & Kivirikko (1987) EMBO J. 6, 643-649]. The procedure involves purification of the prolyl 4-hydroxylase tetramer by a simple affinity chromatography and subsequent isolation of the beta-subunit from the dissociated tetramer by ion-exchange chromatography.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

A simple procedure for the manual isolation and identification of plant heterokaryons

A simple procedure is described for the aseptic manual isolation of individual heterokaryons. Heterokaryons were identified with bright field illumination using an inverted microscope and isolated by means of a micro-manipulator and capillary pipette coupled to a specially constructed syringe. When cell suspension protoplasts were labelled with fluorescein isothiocyanate and fused with mesophyll protoplasts, the heterokaryons exhibited an apple green cytoplasmic fluorescence (from cell suspension protoplasts) and a red chloroplast fluorescence (from mesophyll protoplasts). By this double fluorescence procedure, these manually isolated heterokaryons, identified initially using brightfield could be confirmed as heterokaryons.     

A rapid procedure for the isolation, identification and estimation of citrinin

A rapid procedure for the isolation, identification, and estimation of citrinin, the antibiotic cum nephrotoxin, has been proposed. The colorimetric procedure proposed for estimation makes use of the phenolic function of the compound. This method was found to be applicable to a wide variety of materials.     

High-performance liquid chromatographic procedure for the simultaneous determination of the natural polyamines and their monoacetyl derivatives

The separation of the natural polyamines and their monoacetyl derivatives by high-performance reversed-phase liquid chromatography is reported. Octane sulfonate was used to form ion pairs with the polycations and the o-phthalaldehyde method for post-column derivatization. The method allows polyamine and acetylspermidine determinations directly from tissue extracts and body fluids without pre-purification.     

A modified procedure for isolation of yeast mitochondrial DNA

A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances.