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Purification Protocol (purification + protocol)
Selected AbstractsA fast and inexpensive DNA extraction/purification protocol for brown macroalgaeMOLECULAR ECOLOGY RESOURCES, Issue 2 2007GALICE HOARAU Abstract Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < ,0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated. [source] Development of a Solid-Phase Extraction,Enzyme-Linked Immunosorbent Assay for the Determination of 17,-19-Nortestosterone Levels in Antifatigue Functional FoodsJOURNAL OF FOOD SCIENCE, Issue 8 2009Yan Zhang ABSTRACT:, 17,-19-nortestosterone (17,-NT) has been illegally used in antifatigue functional foods to promote muscle growth and improve endurance. A rapid and sensitive solid-phase extraction,enzyme-linked immunosorbent assay (SPE-ELISA) method was developed and successfully applied to analyze the levels of 17,-NT in antifatigue functional foods. A polyclonal antibody against 17,-NT was produced from rabbits immunized with the 17,-NT-BSA conjugate, and a competitive direct enzyme-linked immunosorbent assay was developed for the rapid detection of 17,-NT. The concentration causing 50% inhibition (IC50) and the limit of detection (LOD) were found to be 0.08 and 0.0055 ng/mL, respectively; this was better than methods previously reported that had a LOD of 2.4 ng/mL. C18 cartridges were investigated for use in removing the effects of matrix in foods, and the sample purification protocol was optimized. Using the developed SPE-ELISA method, recoveries of functional food samples were obtained in the range of 71% to 91.5%. Moreover, 2 kinds of antifatigue functional foods were analyzed using the established ELISA and HPLC methods. The correlation coefficient of the results obtained using the 2 methods was greater than 0.98. Thus, the preliminary evaluation of the SPE-ELISA method proved that it is a specific, sensitive, and precise tool that can be used for the practical detection of 17,-NT in various antifatigue functional food samples. [source] Purification and biochemical characterisation of a novel glutamate decarboxylase from rice branJOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 6 2010Li Wang Abstract BACKGROUND: Glutamate decarboxylase (GAD) is a useful enzyme whose main function is to catalyse the irreversible ,-decarboxylation of L -glutamate to produce ,-aminobutyric acid. The cheap and abundant rice-processing by-product rice bran contains a high amount of GAD, the purification and characterisation of which have not yet been reported. In this study, research on rice bran GAD was initiated. RESULTS: Rice bran GAD was purified to homogeneity via a combined purification protocol of ammonium sulfate fractionation, ion exchange chromatography and two gel filtrations, with a purification fold of 128.6 and an activity recovery of 21.3%. The enzyme was active at pH 5.5 and 40 °C and retained 80% of its original activity in the pH range 5,9 and the temperature range 30,50 °C. GAD activity was significantly enhanced in the presence of Ca2+ but strongly inhibited by Ag+, Hg2+, sodium dodecyl sulfate and CH3COOH. Kinetic determination of the apparent Km for L -glutamate and pyridoxal 5,-phosphate gave values of 27.4 mmol L,1 and 1.16 µmol L,1 respectively. CONCLUSION: Considering that rice bran is cheap and commercially available and that rice bran GAD is relatively stable, the development of cost-effective rice bran GAD-related functional foods would seem to be feasible. Copyright © 2010 Society of Chemical Industry [source] A synergistic approach to protein crystallization: Combination of a fixed-arm carrier with surface entropy reductionPROTEIN SCIENCE, Issue 5 2010Andrea F. Moon Abstract Protein crystallographers are often confronted with recalcitrant proteins not readily crystallizable, or which crystallize in problematic forms. A variety of techniques have been used to surmount such obstacles: crystallization using carrier proteins or antibody complexes, chemical modification, surface entropy reduction, proteolytic digestion, and additive screening. Here we present a synergistic approach for successful crystallization of proteins that do not form diffraction quality crystals using conventional methods. This approach combines favorable aspects of carrier-driven crystallization with surface entropy reduction. We have generated a series of maltose binding protein (MBP) fusion constructs containing different surface mutations designed to reduce surface entropy and encourage crystal lattice formation. The MBP advantageously increases protein expression and solubility, and provides a streamlined purification protocol. Using this technique, we have successfully solved the structures of three unrelated proteins that were previously unattainable. This crystallization technique represents a valuable rescue strategy for protein structure solution when conventional methods fail. [source] Computer-aided NMR assay for detecting natively folded structural domains,PROTEIN SCIENCE, Issue 4 2006Takayuki Hondoh Abstract Structural genomics projects require strategies for rapidly recognizing protein sequences appropriate for routine structure determination. For large proteins, this strategy includes the dissection of proteins into structural domains that form stable native structures. However, protein dissection essentially remains an empirical and often a tedious process. Here, we describe a simple strategy for rapidly identifying structural domains and assessing their structures. This approach combines the computational prediction of sequence regions corresponding to putative domains with an experimental assessment of their structures and stabilities by NMR and biochemical methods. We tested this approach with nine putative domains predicted from a set of 108 Thermus thermophilus HB8 sequences using PASS, a domain prediction program we previously reported. To facilitate the experimental assessment of the domain structures, we developed a generic 6-hour His-tag-based purification protocol, which enables the sample quality evaluation of a putative structural domain in a single day. As a result, we observed that half of the predicted structural domains were indeed natively folded, as judged by their HSQC spectra. Furthermore, two of the natively folded domains were novel, without related sequences classified in the Pfam and SMART databases, which is a significant result with regard to the ability of structural genomics projects to uniformly cover the protein fold space. [source] On the routine use of soft X-rays in macromolecular crystallography.ACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2007Part IV. 23 different crystal forms of 19 different biological macromolecules were examined with respect to their anomalously scattering substructures using diffraction data collected at a wavelength of 2.0,Å (6.2,keV). In more than 90% of the cases the substructure was found to contain more than just the protein S atoms. The data presented suggest that chloride, sulfate, phosphate or metal ions from the buffer or even from the purification protocol are frequently bound to the protein molecule and that these ions are often overlooked, especially if they are not bound at full occupancy. Thus, in order to fully describe the macromolecule under study, it seems desirable that any structure determination be complemented with a long-wavelength data set. [source] Crystallization of the 43,kDa ATPase domain of Thermus thermophilus gyrase B in complex with novobiocinACTA CRYSTALLOGRAPHICA SECTION D, Issue 8 2002V. Lamour The 43,kDa ATPase domain of Thermus thermophilus gyrase B was overproduced in Escherichia coli and a three-step purification protocol yielded large quantities of highly purified enzyme which remained stable for weeks. Crystals of the 43,kDa domain in complex with novobiocin, one of the most potent inhibitors of bacterial topoisomerases, were obtained. Crystals obtained in the presence of PEG 8000 do not diffract, but a different crystal form was obtained using sodium formate as a precipitating agent. The plate-shaped crystals, which were less than 10,µm in thickness, could be cryocooled directly from the mother liquor and a full diffraction data set was collected to 2.3,Å allowing the determination of the first structure of a gyrase B 43K domain in complex with a coumarin. [source] Application of a PEG/salt aqueous two-phase partition system for the recovery of monoclonal antibodies from unclarified transgenic tobacco extractBIOTECHNOLOGY JOURNAL, Issue 9 2009Dimitris Platis Abstract Aqueous two-phase partition systems (ATPS) have been widely used for the separation of a large variety of biomolecules. In the present report, the application of a polyethylene glycol/phosphate (PEG/phosphate) ATPS for the separation of anti-HIV monoclonal antibodies 2G12 (mAb 2G12) and 4E10 (mAb 4E10) from unclarified transgenic tobacco crude extract was investigated. Optimal conditions that favor opposite phase partitioning of plant debris/mAb as well as high recovery and purification were found to be 13.1% w/w (PEG 1500), 12.5% w/w (phosphate) at pH 5 with a phase ratio of 1.3 and 8.25% w/w unclarified tobacco extract load. Under these conditions, mAb 2G12 and mAb 4E10 were partitioned at the bottom phosphate phase with 85 and 84% yield and 2.4- and 2.1-fold purification, respectively. The proposed ATPS was successfully integrated in an affinity-based purification protocol, using Protein A, yielding antibodies of high purity and yield. In this study, ATPS was shown to be suitable for initial protein recovery and partial purification of mAb from unclarified transgenic tobacco crude extract. [source] Recombinant murine growth hormone from E. coli inclusion bodies: Expression, high-pressure solubilization and refolding, and characterization of activity and structureBIOTECHNOLOGY PROGRESS, Issue 3 2010Amber Haynes Fradkin Abstract We expressed recombinant murine growth hormone (rmGH) in E. coli as a cost-effective way to produce large quantities (gram scale) of the protein for use in murine studies of immunogenicity to therapeutic proteins. High hydrostatic pressure was used to achieve high solubility and high refolding yields of rmGH protein produced in E. coli inclusion bodies. A two-step column purification protocol was used to produce 99% pure monomeric rmGH. Secondary and tertiary structures of purified rmGH were investigated using circular dichroism and 2D-UV spectroscopy. The purified rmGH produced was found to be biologically active in hypophysectomized rats. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Contrast analysis of the composition of ribosomes extracted with different purification proceduresJOURNAL OF APPLIED CRYSTALLOGRAPHY, Issue 4 2000Giuseppe Briganti The composition and hydration of E. coli ribosomes isolated with different purification protocols has been analysed by combining two experimental techniques: measurements of small-angle neutron scattering (SANS), for two different isotopic solvent compositions, and refractive index (RI) increments. From the contrast between the solvent and solute scattering densities and the molar polarizability, determined experimentally with SANS and RI measurements, three independent equations are obtained and three unknown quantities are determined: (i) the volume of the solute hydrated skeleton Vs, (ii) the material contained in it, namely the biological components, intrinsic (rRNA and proteins) and extrinsic, such as aminoacylsynthetase and elongation factors, (iii) the number of water molecules structurally bound to the ribosome and non-exchangeable with the solvent. From the form factor at infinite contrast, a second definition of the solute volume is obtained, , which represents the volume within the contour surface of the ribosome. This value is generally larger than Vs and can include a certain amount of water molecules, i.e. those inside the volume (,Vs). Considering the molar volume of this water to be equal to that of the bulk water, it is possible to evaluate its amount. The particle density calculated from the ribosome components in , including proteins, RNA, bound and unbound water molecules, corresponds to the buoyant density measured for E. coli 70S particles. The two ribosomal preparations display different performances in protein synthesis; hence the results indicate that the optimal condition corresponds to a wider skeleton and contour volume but containing a smaller amount of segregated water molecules. It is believed that the method provides a reliable technique to determine the composition of ribosomes under various experimental conditions. [source] High-resolution diffracting crystals of intrinsically active p38, MAP kinase: a case study for low-throughput approachesACTA CRYSTALLOGRAPHICA SECTION D, Issue 2 2007Ron Diskin p38 MAP kinases are central signalling molecules that mediate cellular responses to numerous environmental conditions and signalling molecules. Their proper function is required for many processes, including stress response, apoptosis, differentiation, growth and even learning and memory. Abnormal activity of p38 MAP kinases is associated with the aetiology of many diseases, making understanding their activation mechanisms highly critical. In this respect, mechanistic insights may be derived from structural studies of recently developed intrinsically active p38, mutants. Unlike wild-type p38,, which routinely crystallized, the active mutants caused severe difficulties during the crystallization process. The main hindrance was found to be protein heterogeneity, which was meticulously resolved by genetically modifying the recombinant protein and optimizing the expression and purification protocols. The success in obtaining crystallizable proteins strongly emphasizes that in certain cases, high-throughput techniques (crystallization robots) together with low-throughput approaches, with careful monitoring and analysis of the results, are essential. [source] | |||||||||||||||||||||||||