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Rapid Protocol (rapid + protocol)
Selected AbstractsA universal and rapid protocol for protein extraction from recalcitrant plant tissues for proteomic analysisELECTROPHORESIS, Issue 13 2006Wei Wang Dr. Abstract A simple and universally applicable protocol for extracting high-quality proteins from recalcitrant plant tissues is described. We have used the protocol with no modification, for a wide range of leaves and fruits. In all cases, this protocol allows to obtain good electrophoretic separation of proteins. As the protocol is rapid, universal, and compatible with silver staining, it could be used for routine protein extraction from recalcitrant plant tissues for proteomic analysis. [source] Improved 2-DE of microorganisms after acidic extractionELECTROPHORESIS, Issue 8 2006Ben R. Herbert Professor Abstract 2-DE separations of protein extracts sometimes have problems with poor resolution and streaking. This problem is particularly apparent with microorganisms, most notably those with a large cell wall. Here we describe a novel, rapid protocol for the extraction of microorganisms in acidic conditions, leading to increased resolution and 2-D gel quality. The efficiency of the protocol is demonstrated with extracts of bacteria, Escherichia coli and Bacillus subtilis; fungus, Trichoderma harzianum and yeast, Saccharomyces cerevisiae. We also demonstrate using a membrane centrifugal filtration, that large acidic molecules in excess of 100,kDa, probably including cell wall material, are responsible for the separation difficulties. A range of acidic extraction conditions were investigated, and it was found that optimal extraction is achieved using an extraction solution acidified to pH,3 by 80,mM citric acid. These findings have significant implications for the proteomic study of many medically, agriculturally and environmentally significant microorganisms, as the cell walls of these organisms are often considerably more complex than many commonly studied laboratory strains. [source] Combining Fluorescent Probes and Biofunctional Magnetic Nanoparticles for Rapid Detection of Bacteria in Human Blood,ADVANCED MATERIALS, Issue 23 2006J. Gao Detection of bacteria in human blood within two hours is achieved through the use of vancomycin-functionalized FePt nanoparticles in combination with a vancomycin-conjugated fluorescent probe, as shown in the figure (B: bacteria; Van: vancomycin; FLA: fluorescein amine). This promises to be a sensitive and rapid protocol for detecting bacteria in blood products or other targets in biological samples. [source] Comparative genomics-guided loop-mediated isothermal amplification for characterization of Pseudomonas syringae pv. phaseolicolaJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2009X. Li Abstract Aims:, To design and evaluate a loop-mediated isothermal amplification (LAMP) protocol by combining comparative genomics and bioinformatics for characterization of Pseudomonas syringae pv. phaseolicola (PSP), the causal agent of halo blight disease of bean (Phaseolus vulgaris L.). Methods and Results:, Genomic sequences of Pseudomonas syringae pathovars, P. fluorescens and P. aeruginosa were analysed using multiple sequence alignment. A pathovar-specific region encoding pathogenicity-related secondary metabolites in the PSP genome was targeted for developing a LAMP assay. The final assay targeted a polyketide synthase gene, and readily differentiated PSP strains from other Pseudomonas syringae pathovars and other Pseudomonas species, as well as other plant pathogenic bacteria, e.g. species of Pectobacterium, Erwinia and Pantoea. Conclusion:, A LAMP assay has been developed for rapid and specific characterization and identification of PSP from other pathovars of P. syringae and other plant-associated bacteria. Significance and Impact of the Study:, This paper describes an approach combining a bioinformatic data mining strategy and comparative genomics with the LAMP technology for characterization and identification of a plant pathogenic bacterium. The LAMP assay could serve as a rapid protocol for microbial identification and detection with significant applications in agriculture and environmental sciences. [source] LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01)JOURNAL OF MEDICAL VIROLOGY, Issue 2 2009Avettand-Fènoël Véronique Abstract HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/106 leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r,=,0.900, P,<,0.0001). A total of 3,002 specimens from 1,135 infants were tested. The specificity of HIV-DNA and HIV-RNA assays was 100%. HIV-1 infection was diagnosed in nine infants before age 60 days. HIV-DNA levels were low, underlining the need for sensitive assays when highly active antiretroviral therapy (HAART) has been given. The performances of this HIV-DNA assay showed that it is adapted to early diagnosis in children. The results were equivalent to those of HIV-RNA assay. HIV-DNA may be used even in masked primary infection in newborns whose mothers have received HAART. J. Med. Virol. 81:217,223, 2009. © 2008 Wiley-Liss, Inc. [source] Rapid purification of active ,-secretase, an intramembrane protease implicated in Alzheimer's diseaseJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Matthias Cacquevel Abstract ,-Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid-, precursor protein generates the amyloid-, protein (A,) of Alzheimer's disease, partially inhibiting ,-secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of ,-secretase, but at low resolution (15 Å). The limited amount of purified ,-secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X-ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S-20) that overexpresses strikingly high levels of all four ,-secretase components (presenilin, nicastrin, Aph-1 and Pen-2). We then used these cells to develop a rapid protocol for the high-grade purification of proteolytically active ,-secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme. [source] Gerbera jamesonii, a New Host of Fusarium oxysporum f.sp. tracheiphilumJOURNAL OF PHYTOPATHOLOGY, Issue 1 2010Marco Troisi Abstract The random amplified polymorphic DNA (RAPD) technique was used to analyze the total genomic DNA of pathogenic isolates of Fusarium oxysporum on Gerbera jamesonii by comparing them to representatives of the formae speciales chrysanthemi and tracheiphilum. A close genetic relationship was observed among most of the new isolates from G. jamesonii. They shared RAPD markers with the tested representatives of the forma specialis chrysanthemi. Some isolates of those tested from diseased G. jamesonii were placed in a different cluster, which included representative isolates of forma specialis tracheiphilum. This is the first report of F. oxysporum f.sp. tracheiphilum on G. jamesonii. A rapid protocol for DNA extraction directly from fungal colonies grown on potato dextrose agar allowed complete analysis in less than 4 h. [source] | ||||||||||