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Extraction Buffer (extraction + buffer)
Selected AbstractsPersistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water,ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2008Hirofumi Kosaki Abstract Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline. [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] 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] Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-ThawingBIOTECHNOLOGY PROGRESS, Issue 6 2006Toru Ezure We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and ,-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5,-untranslated region (5,-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5,-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5,-UTRs tested. As a result, in a batch reaction approximately 71 ,g of luciferase was synthesized per milliliter of reaction volume at 25 °C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5,-UTR of mRNA, approximately 45 ,g/mL of luciferase was synthesized in an Sf21 cell-free system at 25 °C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method. [source] Abundant Tissue Butyrylcholinesterase and Its Possible Function in the Acetylcholinesterase Knockout MouseJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Bin Li Abstract: We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well. [source] A high-throughput protocol for extracting high-purity genomic DNA from plants and animalsMOLECULAR ECOLOGY RESOURCES, Issue 4 2008R. WHITLOCK Abstract DNA extraction techniques that employ the reversible binding of DNA to silica via chaotropic salts can deliver high-quality genomic DNA from plant and animal tissues, while avoiding the use of toxic organic solvents. Existing techniques that use this method are either prohibitively expensive, or are applicable to only a restricted set of taxa. Here we describe a cost-effective DNA extraction technique suitable for a wide range of plant and animal taxa that yields microgram quantities of high-molecular-weight genomic DNA at a throughput of 192 samples per day. Our technique is particularly robust for tissue samples that are insoluble or are rapidly discoloured or oxidized in standard DNA extraction buffers. We demonstrate the quality of DNA extracted using this method by applying the amplified fragment length polymorphism technique to plant species. [source] Carbonate extraction process for the metabolic, isozymic and proteomic profiling of rose-scented geranium (Pelargonium sp.), a hyper-acidic plantPHYTOCHEMICAL ANALYSIS, Issue 2 2008Rajender Singh Sangwan Abstract Rose-scented geranium (Pelargonium sp.) is a valuable monoterpene-yielding plant. It has been well characterised phytochemically through the isolation of >270 secondary metabolites, however, there is hardly any biochemical or metabolic information concerning this plant. Initial attempts to investigate its metabolism failed to produce any enzyme activity in the tissue extracts prepared in routine extraction buffers owing to the intrinsic properties of the tissue matrix. It was recognised that cellular hyper-acidity (cell sap pH ,3.0) gave rise to very low protein levels in the extracts, thus prohibiting detection of activities of even primary metabolic enzymes that are usually abundantly present in plants. Tissue extraction in Tris solution without pH adjustment (as used for studies involving citrus and banana) led to little or no improvement. Therefore, a novel approach using sodium carbonate solution as an efficient extraction system for enzymes and proteins from the plant was studied. Functionality of the carbonate extraction has been demonstrated through its effectiveness, a several-fold superior performance, in yielding protein, monitoring primary metabolism and secondary metabolic enzymes, and isozymic and polypeptide profiling. The process may also be helpful in the reliable analysis of other acidic plant tissues. Copyright © 2007 John Wiley & Sons, Ltd. [source] | ||||||||||