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Extraction Protocol (extraction + protocol)
Kinds of Extraction Protocol Selected AbstractsMass spectrometric characterization of the covalent modification of the nitrogenase Fe-protein in Azoarcus sp.FEBS JOURNAL, Issue 13 2009Nitrogenase Fe-protein modification was analyzed in the endophytic ,-proteobacterium Azoarcus sp. BH72. Application of modern MS techniques localized the modification in the peptide sequence and revealed it to be an ADP-ribosylation on Arg102 of one subunit of nitrogenase Fe-protein. A double digest with trypsin and endoproteinase Asp-N was necessary to obtain an analyzable peptide because the modification blocked the trypsin cleavage site at this residue. Furthermore, a peptide extraction protocol without trifluoroacetic acid was crucial to acquire the modified peptide, indicating an acid lability of the ADP-ribosylation. This finding was supported by the presence of a truncated version of the original peptide with Arg102 exchanged by ornithine. Site-directed mutagenesis verified that the ADP-ribosylation occurred on Arg102. With our approach, we were able to localize a labile modification within a large peptide of 31 amino acid residues. The present study provides a method suitable for the identification of so far unknown protein modifications on nitrogenases or other proteins. It represents a new tool for the MS analysis of protein mono-ADP-ribosylations. [source] DNA Extraction from Olive Oil and PCR Amplification of Microsatellite MarkersJOURNAL OF FOOD SCIENCE, Issue 1 2005Raffaele Testolin And ABSTRACT: DNA was extracted from single-cultivar of cold-pressed (virgin) unfiltered and cotton-filtered olive oils that were stored at 4 °C for up to a year using different DNA extraction kits and protocols. DNA was amplified using original and nested primers designed on 6 microsatellites loci of the UDO series. The most consistent results in terms of successful single sequence repeat amplifications were achieved using the Qiagen QIAamp DNA stool extraction kit, slightly modified and applied to oil sample amounts as small as 200 ,L without any pretreatment. The kit allowed getting polymerase chain reaction (PCR) amplicons visible on gel and scorable peaks at the automatic sequencer for all 6 markers analyzed. Less consistent results were achieved with other kits, such as the Promega Wizard Magnetic DNA Purification System for Food, the LB Link-Biotech ExtMan 50,100 Evolution, the Qiagen Plant Mini kit, and the standard cetyltrimethyl-ammonium bromide-based DNA extraction protocol. The integration in the protocols of further tools, such as the hexane-based phase separation, the addition of water or NaCl solutions to the oil, the precipitation and the use of the pellet, and others, did not result in any substantial use. PCR amplifications that gave low DNA yields were improved by adopting the nested PCR technique, which uses the product of the 1st PCR as a template for a 2nd PCR carried out by means of internal primers. Conclusions are drawn as to the applicability of the method to trace the identity of single-cultivar virgin olive oils. Further work is required to check the sensitivity of the method in determining the varietal composition of blended oils, especially in detecting alleles from cultivars present in only small amounts. [source] Determination of banned Sudan dyes in food samples by molecularly imprinted solid phase extraction-high performance liquid chromatographyJOURNAL OF SEPARATION SCIENCE, JSS, Issue 19 2009Claudio Baggiani Abstract A method for molecularly imprinted SPE of banned Sudan azo-dyes from food samples was investigated. The molecularly imprinted polymer was obtained by suspension polymerization using 1-(4-chlorophenyl)azonaphthalen-2-ol as the mimic template. The molecular recognition properties of imprinted beads were evaluated for use as a SPE sorbent, in order to develop a selective extraction protocol for the Sudan class of dyes. The optimized extraction protocol resulted in a reliable molecularly imprinted SPE (MISPE) method suitable for HPLC analysis. It was selective for the main analyte, Sudan I, and the related azo-dyes Sudan II, III, IV, Sudan Red B, and Sudan Red 7B, while the permitted azo-dyes Allura Red AC, Neococcin, and Sunset Yellow FCF were not extracted. The method was tested for Sudan I, II, III, and IV in five different food samples (hot chilli pepper, hot chilli tomato sauce, sausage, tomato sauce, and hard boiled egg yolk) at three concentration levels (15, 100, and 300 ,g/g). It demonstrated itself to be insensitive to the presence of different complex matrices, precise, accurate, and with good recovery rates (85,101%). The LOD and LOQ were satisfactory for most analytical determinations. [source] Single sample extraction protocol for the quantification of NAD and NADH redox states in Saccharomyces cerevisiaeJOURNAL OF SEPARATION SCIENCE, JSS, Issue 18 2008Jennifer L. Sporty Abstract A robust redox extraction protocol for quantitative and reproducible metabolite isolation and recovery has been developed for simultaneous measurement of nicotinamide adenine dinucleotide (NAD) and its reduced form, NADH, from Saccharomyces cerevisiae. Following culture in liquid media, yeast cells were harvested by centrifugation and then lysed under nonoxidizing conditions by bead blasting in ice-cold, nitrogen-saturated 50 mM ammonium acetate. To enable protein denaturation, ice cold nitrogen-saturated CH3CN/50 mM ammonium acetate (3:1 v/v) was added to the cell lysates. Chloroform extractions were performed on supernatants to remove organic solvent. Samples were lyophilized and resuspended in 50 mM ammonium acetate. NAD and NADH were separated by HPLC and quantified using UV,Vis absorbance detection. NAD and NADH levels were evaluated in yeast grown under normal (2% glucose) and calorie restricted (0.5% glucose) conditions. Results demonstrate that it is possible to perform a single preparation to reliably and robustly quantitate both NAD and NADH contents in the same sample. Robustness of the protocol suggests it will be (i) applicable to quantification of these metabolites in other cell cultures; and (ii) amenable to isotope labeling strategies to determine the relative contribution of specific metabolic pathways to total NAD and NADH levels in cell cultures. [source] DNA extraction method for PCR in mycorrhizal fungiLETTERS IN APPLIED MICROBIOLOGY, Issue 4 2001S. Manian Aims: To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. Methods and Results: The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. Conclusions: DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. Significance and Impact of the Study: The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi. [source] Vouchering DNA-barcoded specimens: test of a nondestructive extraction protocol for terrestrial arthropodsMOLECULAR ECOLOGY RESOURCES, Issue 6 2007DANIEL L. ROWLEY Abstract Morphology-based keys support accurate identification of many taxa. However, identification can be difficult for taxa that are either not well studied, very small, members of cryptic species complexes, or represented by immature stages. For such cases, DNA barcodes may provide diagnostic characters. Ecologists and evolutionary biologists deposit museum vouchers to document the species studied in their research. If DNA barcodes are to be used for identification, then both the DNA and the specimen from which it was extracted should be vouchered. We describe a protocol for the nondestructive extraction of DNA from terrestrial arthropods, using as examples members of the orders Acarina, Araneae, Coleoptera, Diptera, and Hymenoptera chosen to represent the ranges in size, overall sclerotization, and delicacy of key morphological characters in the group. We successfully extracted sequenceable DNA from all species after 1,4 h of immersion in extraction buffer. The extracted carcasses, processed and imaged using protocols standard for the taxon, were distinguishable from closely related species, and adequate as morphological vouchers. We provide links from the carcasses and DNA vouchers to image (MorphBank) and sequence (GenBank) databases. [source] An effective DNA extraction protocol for brown algaePHYCOLOGICAL RESEARCH, Issue 2 2001Naomi Phillips SUMMARY Successful extraction of total DNA from brown algae, which are generally polysaccharide and polyphenol rich, is often problematic using current methods. Persistent polysaccharide and polyphenolic compounds can hinder further application of modern molecular techniques requisite to molecular-based evolutionary studies. Our broad and long-term research goals with fucalean taxa, especially Sargassum, and problems with existing DNA extraction methods were an impetus to develop a reliable DNA extraction method. Initial research established hexadecyltrimethylammonium bromide (CTAB) based total-DNA methods as the most viable for further empirical development. Several constituents effective at either complexing secondary compounds or creating a reductive extraction environment were increased in concentration or added to the extraction buffer. These seemingly minor changes resulted in the creation of a highly reductive extraction buffer and effective total- DNA harvesting technique. We detail these modifications and demonstrate the reliability of the modified protocol with a variety of brown algae and tissue preservation methods. Such DNA is shown to be suitable for a variety of molecular techniques. [source] Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene familyPHYTOCHEMICAL ANALYSIS, Issue 4 2003Nathalie Mattheus Abstract Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280,nm and 260/230,nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5,µg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picg1, that was very similar to members of the PR10 gene family. Copyright © 2003 John Wiley & Sons, Ltd. [source] Improved chromatographic fingerprints for facile differentiation of two Ganoderma spp.BIOMEDICAL CHROMATOGRAPHY, Issue 3 2009Chun-Mei Fu Abstract This paper addresses a comprehensive and comparative study of six phytochemical extraction methods for triterpenes from the fruiting body of Ganoderma spp. Quantitative analysis of extracts was performed by HPLC with photodiode array detection. In general, pressurized liquid extraction and microwave-assisted extraction under optimized conditions produce better yields, and the former also significantly reduces the total time of extraction and manipulation of a sample, as well as the amount of solvent used in comparison with conventional soxhlet, reflux, ultrasonic, and methanol,CO2 supercritical fluid extractions. Based on the improved extraction protocol, the fingerprinting profiles for two species of Lingzhi were established using the consistent chromatographic features of 12 authentic samples. Eleven common peaks of ganoderic/ganoderenic acids were identified using LC-ESI-MS-MS. These specific triterpene groups were adopted as chemical markers for Lingzhi. Using chemometric analysis, the developed fingerprinting was successfully applied to differentiate between the two species under the Ganoderma genus and is applicable as a method for quality evaluation of this valuable medicinal fungus and its related proprietary products. Copyright © 2008 John Wiley & Sons, Ltd. [source] Methods for detecting and identifying retinoids in tissueDEVELOPMENTAL NEUROBIOLOGY, Issue 7 2006Thomas E. Gundersen Abstract Methods for retinoid analysis in tissue include direct spectrophotometry or fluorometry and retinoid responsive reporter constructs in the form of cell reporter assays or transgenic reporter animals, but chromatographic methods dominate and posses several superior features in quantitative analysis. The multitude of extraction protocols used can coarsely be divided into manual liquid-liquid extraction protocols and semi- or fully automated solid phase extraction-based protocols. Liquid chromatographic separation in reversed phase dominates although normal phase is also used. Detection is mainly performed with UV detectors although electrochemical and fluorescence detection is also used. Mass spectrometry in combination with LC is more often used in retinoid analysis and is likely to dominate in the future. © 2006 Wiley Periodicals, Inc. J Neurobiol 66: 631,644, 2006 [source] Proteomics in globe artichoke: Protein extraction and sample complexity reduction by PEG fractionationELECTROPHORESIS, Issue 9 2009Alberto Acquadro Abstract Here, we report the first leaf proteome analysis for globe artichoke. Three protein extraction protocols were tested and a reproducible Mg/NP-40-based method was established. Ribulose-1,5-biphosphate carboxylase-oxygenase (RuBisCO) is a highly abundant leaf protein, and its presence masks co-localizing, less abundant proteins. To remove RuBisCO from the sample, and thereby improve spot resolution, a PEG fractionation approach was elaborated. 2-DE profiles of various PEG fractions showed that the fractionation procedure was successful in excluding most of the RuBisCO, allowing for the detection of many low-abundance proteins. Western blot analysis was able to confirm the reduction in RuBisCO content achieved by PEG fractionation. In all, 841 distinct protein spots were detected, and 40 of these, selected from the RuBisCO region of the 2-DE profile, were successfully identified by MS. A number of homologues of these proteins also co-localize with RuBisCO in Arabidopsis thaliana. [source] Proteomic analysis of RCL2 paraffin-embedded tissuesJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 5b 2008V. Belief Abstract Histopathological diagnosis in most of the world's hospitals is based upon formalin-fixed and paraffin-embedded (FFPE) tissues. Although this standard fixation and embedding procedure keeps the tissue in excellent form for morphological and immunohistological analysis, FFPE is inappropriate for nucleic acids and protein studies. We investigated the potential value of RCL2, a new non-toxic fixative, for sparing proteins preserved in paraffin-embedded tissues. Normal colonic mucosa tissue was fixed in RCL2 prior to paraffin embedding (RCL2P), and then processed for quality and quantity of protein conservation, as compared to frozen and FFPE tissues using complementary proteomic analysis approaches. Using 4 different protein extraction protocols, RCL2P tissue consistently showed the highest protein yield. Similar protein patterns were observed with RCL2P and frozen tissues using mono and bi-dimensional electrophoresis. Moreover, membrane, cytoplasmic and nuclear proteins, as well as phosphorylated proteins, were successfully detected using western-blot. Furthermore, protein patterns observed by mass spectrometry analysis after laser-captured microdissection were found to be identical for frozen and RCL2-fixed tissues. At last, immunohistochemistry using various antibodies showed comparable results between both tissue storage methods. We concluded that RCL2 has great potential for performing both morphological and molecular analyses on the same archival paraffin-embedded tissue sample, and can be a new method for investigating protein biomarkers. [source] MALDI-TOF mass spectrometry of hordeins: rapid approach for identification of malting barley varietiesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 9 2009alplachta Abstract A procedure for identification of malting barley varieties using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of ethanol-soluble barley proteins (hordeins) is described. The hordeins were first extracted from milled barley grains by several extraction protocols (using different extraction agents and conditions). Hordein extracts were then analyzed directly via MALDI-TOF MS without any preliminary purification or separation step, and the protein profiles of analyzed hordein extracts were compared in order to find out the most suitable extraction procedure for mass spectrometric analysis. The optimized procedure was successfully applied to identification of 13 malting barley varieties. Our results revealed that the proposed mass spectrometry-based approach provides characteristic mass patterns of extracted hordeins, which can be advantageously used for barley variety identification. Copyright © 2009 John Wiley & Sons, Ltd. [source] The use of biomagnetic separation to recover DNA suitable for PCR from Claviceps species ,LETTERS IN APPLIED MICROBIOLOGY, Issue 2 2000D.L. Scott Jr DNA analysis of agriculturally important fungi using polymerase chain reaction (PCR)-based methods is becoming routine in research and for diagnostic purposes. Rapid, small-scale DNA isolation methods that take advantage of the sensitivity, speed and automation potential of PCR technology are needed for timely analysis of important plant pathogens. DNA isolated from Claviceps africana (causal agent of ergot of sorghum) using several standard DNA extraction protocols was found to be unamplifiable using PCR. The standard methods apparently failed to separate DNA from substances inhibitory to the Taq polymerase enzyme. We obtained DNA amenable to PCR analysis using a novel method involving magnetic beads and high salt extraction buffer. The biomagnetic purification method allowed us to obtain reliable PCR amplification of the internal transcribed spacer (ITS) regions of rDNA of Claviceps africana, making genetic comparisons possible. [source] Isolation of high-quality RNA from white spruce tissue using a three-stage purification method and subsequent cloning of a transcript from the PR-10 gene familyPHYTOCHEMICAL ANALYSIS, Issue 4 2003Nathalie Mattheus Abstract Isolation of PinmIII cDNA homologues from white spruce tissues required a rigorous RNA extraction protocol developed following assessment of three previously reported conifer RNA extraction protocols. Total RNA was extracted via several purification steps designed to minimize binding of phenolics to nucleic acids and was then subjected to caesium chloride ultra-centrifugation. This procedure produced consistently high-quality, intact RNA from both needles and roots with spectrophotometric ratios of approximately 2.0 for both 260/280,nm and 260/230,nm. Total RNA was obtained from the roots of cold-hardened white spruce seedlings for cDNA library construction. More than 2 million recombinant phage particles were generated from 5,µg of a poly(A)+RNA fraction, and ca. 1.3 million cDNA particles were amplified for storage. Approximately 500,000 primary recombinant clones were screened with an heterologous PinmIII cDNA sequence yielding a unique clone, picg1, that was very similar to members of the PR10 gene family. Copyright © 2003 John Wiley & Sons, Ltd. [source] Rapid screening and confirmation of drugs and toxic compounds in biological specimens using liquid chromatography/ion trap tandem mass spectrometry and automated library searchRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 1 2010Hsiu-Chuan Liu Recent advances in liquid chromatography/tandem mass spectrometry (LC/MS/MS) technology have provided an opportunity for the development of more specific approaches to achieve the ,screen' and ,confirmation' goals in a single analytical step. For this purpose, this study adapts the electrospray ionization ion trap LC/MS/MS instrumentation (LC/ESI-MS/MS) for the screening and confirmation of over 800 drugs and toxic compounds in biological specimens. Liquid-liquid and solid-phase extraction protocols were coupled to LC/ESI-MS/MS using a 1.8-µm particle size analytical column operated at 50°C. Gradient elution of the analytes was conducted using a solvent system composed of methanol and water containing 0.1% formic acid. Positive-ion ESI-MS/MS spectra and retention times for each of the 800 drugs and toxic compounds were first established using 1,10,µg/mL standard solutions. This spectra and retention time information was then transferred to the library and searched by the identification algorithm for the confirmation of compounds found in test specimens , based on retention time matches and scores of fit, reverse fit, and purity resulting from the searching process. The established method was found highly effective when applied to the analyses of postmortem specimens (blood, urine, and hair) and external proficiency test samples provided by the College of American Pathology (CAP). The development of this approach has significantly improved the efficiency of our routine laboratory operation that was based on a two-step (immunoassay and GC/MS) approach in the past. Copyright © 2009 John Wiley & Sons, Ltd. [source] Determination of nitrogen-15 isotope fractionation in tropine: evaluation of extraction protocols for isotope ratio measurement by isotope ratio mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2009Roland Molinié N -Demethylation of tropine is an important step in the degradation of this compound and related metabolites. With the purpose of understanding the reaction mechanism(s) involved, it is desirable to measure the 15N kinetic isotope effects (KIEs), which can be accessed through the 15N isotope shift (,,15N) during the reaction. To measure the isotope fractionation in 15N during tropine degradation necessitates the extraction of the residual substrate from dilute aqueous solution without introducing artefactual isotope fractionation. Three protocols have been compared for the extraction and measurement of the 15N/14N ratio of tropine from aqueous medium, involving liquid-liquid phase partitioning or silica-C18 solid-phase extraction. Quantification was by gas chromatography (GC) on the recovered organic phase and ,15N values were obtained by isotope ratio measurement mass spectrometry (irm-MS). Although all the protocols used can provide satisfactory data and both irm-EA-MS and irm-GC-MS can be used to obtain the ,15N values, the most convenient method is liquid-liquid extraction from a reduced aqueous volume combined with irm-GC-MS. The protocols are applied to the measurement of 15N isotope shifts during growth of a Pseudomonas strain that uses tropane alkaloids as sole source of carbon and nitrogen. The accuracy of the determination of the 15N/14N ratio is sufficient to be used for the determination of 15N-KIEs. Copyright © 2009 John Wiley & Sons, Ltd. [source] | |||||||||||||