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Isolation Protocols (isolation + protocol)
Selected AbstractsIsolation and purification of macrocyclic components from Penicillium fermentation broth by high-speed counter-current chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 2 2010Xiang Gao Abstract In this paper, high-speed counter-current chromatography (HSCCC), assisted with ESI-MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6,mg, 99.0%), 7,- O -formylbrefeldin A (6.5,mg, 95.0%) and 7,- O -acetylbrefeldin A (5.0,mg, 92.3%) from the crude extract of the microbe Penicillium SHZK-15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two-step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two-step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n -hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5,v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5,v/v/v/v) and HEMWat (7:3:5:5,v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X-ray crystallography, ESI-MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes. [source] Evidence for cyanophages active against bloom-forming freshwater cyanobacteriaFRESHWATER BIOLOGY, Issue 6 2008LI DENG Summary 1. A total of 35 putative cyanophages able to infect non-axenic cultures of bloom-forming freshwater cyanobacteria in the genera Microcystis, Anabaena and Planktothrix were isolated from Lake Zurich (Switzerland) and lakes in the Cotswold Water Park (U.K.). Eleven lytic cyanophage isolates were isolated on Microcystis and 12 each on Anabaena and Planktothrix. Cyanophage isolation protocols varied when using these different cyanobacterial hosts. 2. The collection of putative cyanophage isolates encompassed a variety of morphotypes, including the first filamentous cyanophage from any environment and the second siphocyanophage reported from fresh water. 3. PCR primer sets for gp20, gp23 and MCP genes, which have been previously found to be conserved in other cyanophages, were used in an attempt to determine genetic diversity among the phage isolates. The failure to obtain specific amplification products from most isolates suggests that the cyanophages isolated in this study were different from those previously characterized from both marine and freshwater environments. 4. Some putative cyanophages within the collection of isolates proved to have a very broad host range and were able to infect Anabaena, Microcystis and Planktothrix. The ability to infect a wide range of host taxa extends the potential reproductive period for lytic propagation, and also has implications for the transfer of genetic information between deeply separated cyanobacterial lineages. [source] Effects of different brush border membrane vesicle isolation protocols on proteomic analysis of Cry1Ac binding proteins from the midgut of Helicoverpa armigeraINSECT SCIENCE, Issue 6 2008Li-Zhen Chen Abstract Brush border membrane vesicles (BBMV) isolated from insect midguts have been widely used to study Cry1A binding proteins. Sample preparation is important in two-dimensional electrophoresis (2-DE), so to determine a suitable BBMV preparation method in Helicoverpa armigera for 2-DE, we compared three published BBMV preparation methods mostly used in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). All methods yielded similar types and numbers of binding proteins, but in different quantities. The Abdul-Rauf and Ellar protocol was the best of the three, but had limitations. Sufficient protein quantity is important for research involving limited numbers of insects, such as studies of insect resistance to Bacillus thuringiensis in the field. Consequently, we integrated the three BBMV isolation methods into a single protocol that yielded high quantities of BBMV proteins from H. armigera larval midguts, which proved suitable for 2-DE analysis. [source] Evaluation of polymerase chain reaction and DNA isolation protocols for detection of genetically modified soybeanINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 10 2007Cibele dos Santos Ferrari First page of article [source] Comparison of three plating media for the isolation of Salmonella from poultry environmental samples in Great Britain using ISO 6579:2002 (Annex D)JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009J.J. Carrique-Mas Abstract Aims:, To evaluate the performance of three Salmonella plating media (Rambach, Xylose Lysine Deoxycholate agar and modified Brilliant Green Agar plus Novobiocin) as part of the ISO 6579: 2002 (Annex D) on poultry environmental samples. Methods and Results:, The samples analysed were those for the European Union Salmonella baseline surveys of laying (N = 3087), broiler (N = 1550), turkey fattening (N = 1540) and turkey breeding (N = 580) flocks for Great Britain. Results were considered separately for Rambach (including and excluding pale orange colonies) and for growth on selective media [Modified semi-solid Rappaport Vassiliadis (MSRV)] after 24 and 48 h of incubation. Overall, Rambach was the most sensitive medium, provided that pale orange colonies were checked. In all cases, an increase in the sensitivity of detection was obtained by plating growth on MSRV after 48 h of incubation. In broilers and laying flocks, the specificity significantly improved when Rambach only was used. Conclusion:, The use of Rambach results in considerable savings compared with the two-plate method prescribed by ISO 6579:2002 (Annex D) without compromising sensitivity. Significance and Impact of the Study:,Salmonella isolation protocols should be reviewed in terms of their efficiency and cost. [source] | |||||||||||||