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Sample records for additional purification step

  1. Effect of additives on the purification of urease

    NASA Astrophysics Data System (ADS)

    Yu, X.; Wang, J.; Ulrich, J.

    2015-12-01

    The effect of additives on the purification of proteins was investigated. The target protein studied here is the enzyme urease. Studies on the purification of urease from jack bean meal were carried out. 32% (v/v) acetone was utilized to extract urease from the jack bean meal. Further purification by crystallization with the addition of 2-mercaptoethanol and EDTA disodium salt dehydrate was carried out. It was found out that the presence of additives can affect the selectivity of the crystallization. Increases in both purity and yield of the urease after crystallization were observed in the presence of additives, which were proven using both SDS-PAGE and activity. Urease crystals with a yield of 69.9% and a purity of 85.1% were obtained in one crystallization step in the presence of additives. Furthermore, the effect of additives on the thermodynamics and kinetics of urease crystallization was studied.

  2. Automated multi-step purification protocol for Angiotensin-I-Converting-Enzyme (ACE).

    PubMed

    Eisele, Thomas; Stressler, Timo; Kranz, Bertolt; Fischer, Lutz

    2012-12-12

    Highly purified proteins are essential for the investigation of the functional and biochemical properties of proteins. The purification of a protein requires several steps, which are often time-consuming. In our study, the Angiotensin-I-Converting-Enzyme (ACE; EC 3.4.15.1) was solubilised from pig lung without additional detergents, which are commonly used, under mild alkaline conditions in a Tris-HCl buffer (50mM, pH 9.0) for 48h. An automation of the ACE purification was performed using a multi-step protocol in less than 8h, resulting in a purified protein with a specific activity of 37Umg(-1) (purification factor 308) and a yield of 23.6%. The automated ACE purification used an ordinary fast-protein-liquid-chromatography (FPLC) system equipped with two additional switching valves. These switching valves were needed for the buffer stream inversion and for the connection of the Superloop™ used for the protein parking. Automated ACE purification was performed using four combined chromatography steps, including two desalting procedures. The purification methods contained two hydrophobic interaction chromatography steps, a Cibacron 3FG-A chromatography step and a strong anion exchange chromatography step. The purified ACE was characterised by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native-PAGE. The estimated monomer size of the purified glycosylated ACE was determined to be ∼175kDa by SDS-PAGE, with the dimeric form at ∼330kDa as characterised by a native PAGE using a novel activity staining protocol. For the activity staining, the tripeptide l-Phe-Gly-Gly was used as the substrate. The ACE cleaved the dipeptide Gly-Gly, releasing the l-Phe to be oxidised with l-amino acid oxidase. Combined with peroxidase and o-dianisidine, the generated H(2)O(2) stained a brown coloured band. This automated purification protocol can be easily adapted to be used with other protein purification tasks. PMID:23217308

  3. One-step purification of phosphinothricin acetyltransferase using reactive dye-affinity chromatography.

    PubMed

    Wang, Cunxi; Lee, Thomas C; Crowley, Kathleen S; Bell, Erin

    2015-01-01

    Reactive dye purification is an affinity purification technique offering unique selectivity and high purification potential. Historically, purification of phosphinothricin acetyltransferase (PAT) has involved several steps of precipitation and column chromatography. Here, we describe a novel purification method that is simple, time-saving, inexpensive, and reproducible. The novel method employs a single chromatography step using a reactive dye resin, Reactive brown 10-agarose. Reactive brown 10 preferentially binds the PAT protein, which can then be specifically released by one of its substrates, acetyl-CoA. Using Reactive brown 10-agarose, PAT protein can be purified to homogeneity from E. coli or plant tissue with high recovery efficiency. PMID:25749943

  4. Improving impurities clearance by amino acids addition to buffer solutions for chromatographic purifications of monoclonal antibodies.

    PubMed

    Ishihara, Takashi; Hosono, Mareto

    2015-07-15

    The performance of amino acids in Protein A affinity chromatography, anion exchange chromatography and cation exchange chromatography for monoclonal antibody purification was investigated. Glycine, threonine, arginine, glutamate, and histidine were used as buffer components in the equilibration, washing, and elution steps of these chromatographies. Improved clearance of impurity, high molecular weight species (HMW) and host cell proteins (HCP) was observed in the purification processes when using the amino acids as base-buffer constituents, additives or eluents compared with that of buffers without these amino acids. In addition, we designed a buffer system in which the mobile phases were composed of only a single amino acid, histidine, and applied it to the above three chromatographies. Effective HMW and HCP clearance was also obtained in this manner. These results suggest that amino acids may enhance impurity clearance during the purification of monoclonal antibodies. PMID:26057847

  5. GST-His purification: a two-step affinity purification protocol yielding full-length purified proteins.

    PubMed

    Maity, Ranjan; Pauty, Joris; Krietsch, Jana; Buisson, Rémi; Genois, Marie-Michelle; Masson, Jean-Yves

    2013-01-01

    Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest. PMID:24193370

  6. Column chromatography as a useful step in purification of diatom pigments.

    PubMed

    Tokarek, Wiktor; Listwan, Stanisław; Pagacz, Joanna; Leśniak, Piotr; Latowski, Dariusz

    2016-01-01

    Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies. PMID:27486920

  7. Two-Step Vapor/Liquid/Solid Purification

    NASA Technical Reports Server (NTRS)

    Holland, L. R.

    1986-01-01

    Vertical distillation system combines in single operation advantages of multiple zone refining with those of distillation. Developed specifically to load Bridgman-Stockbarger (vertical-solidification) growth ampoules with ultrapure tellurium and cadmium, system, with suitable modifications, serves as material refiner. In first phase of purification process, ampoule heated to drive off absorbed volatiles. Second phase, evaporator heated to drive off volatiles in charge. Third phase, slowly descending heater causes distillation from evaporator to growing crystal in ampoule.

  8. [A simple and effective step in the purification of bovine blood fibronectin].

    PubMed

    Zykova, T A; Zlatopol'skiĭ, A D; Mazurov, V I

    1983-01-01

    Preparations of fibronectin from bovine blood serum, obtained by means of affinity chromatography on collagen-Sepharose, contained immunoglobulins and other proteins, concentration of which constituted 48 +/- 5%. Differential salting out of fibronectin and other non-fibronectin proteins, using 0.8-2.0 M ammonium sulfate at pH 5.0, demonstrated that precipitation of fibronectin occurred more effectively as compared with non-fibronectin proteins at all the salt concentrations studied. If 0.8 M or 1.0 M ammonium sulfate concentrations were used, the fibronectin preparations contained less than 10% of other proteins and fibronectin loss was about 20%. Salting out of fibronectin is an effective additional step of its purification. PMID:6649521

  9. Host cell protein impurities in chromatographic polishing steps for monoclonal antibody purification.

    PubMed

    Levy, Nicholas E; Valente, Kristin N; Lee, Kelvin H; Lenhoff, Abraham M

    2016-06-01

    Downstream purification of monoclonal antibodies (mAbs) is normally performed using a platform process that is empirically tuned to optimize impurity removal for each new product. A more fundamental understanding of impurities and the product itself would provide insights into the rational design of efficient downstream processes. This work examines the chromatographic properties of Chinese hamster ovary host cell protein (HCP) impurities in non-affinity chromatographic resins commonly used in polishing steps for monoclonal antibody purification: ion-exchange, hydrophobic interaction, and multimodal. Using proteomic analysis, the specific HCP impurities that elute close to mAb products are identified for these resins at typical downstream processing conditions. Additionally, the interactions of HCP impurities with mAb products are profiled to determine the total extent of product association and the specific HCP species that form associative complexes under conditions encountered in polishing columns. Product association and co-elution were both identified as viable mechanisms of HCP retention for the non-affinity resins tested here. A relatively large sub-population of HCP impurities was found to co-elute or associate with mAbs in each polishing column, but only a small population of HCPs-including lipoprotein lipase, chrondroitin sulfate proteoglycan 4, nidogen-1, and SPARC-were identified as difficult to remove across an entire downstream mAb process. Biotechnol. Bioeng. 2016;113: 1260-1272. © 2015 Wiley Periodicals, Inc. PMID:26550778

  10. The Amicon Pro system--a centrifugal device capable of performing all steps in the protein purification workflow.

    PubMed

    Cappione, Amedeo; Mabuchi, Masaharu; Suhrawardy, Saosan; Briggs, David; Nadler, Timothy

    2013-01-01

    raditional protein purification is a long process with many steps utilizing multiple devices, often resulting in protein degradation and loss. The Amicon Pro device streamlines the affinity purification process by providing a single adaptable centrifugation unit capable of performing all steps in the affinity purification process. The device combines affinity-based spin column purification with downstream sample concentration and buffer exchange, eliminating the need for multiple sample transfers, thereby minimizing protein loss. The results presented in this work indicate that purification of His-tagged protein using the Amicon Pro device is faster, easier, and provides better yields than other traditional methods (eg. spin-column and slurry method). PMID:24364216

  11. Optimization of conditions for the single step IMAC purification of miraculin from Synsepalum dulcificum.

    PubMed

    He, Zuxing; Tan, Joo Shun; Lai, Oi Ming; Ariff, Arbakariya B

    2015-08-15

    In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum. PMID:25794715

  12. One-step deterministic polarization-entanglement purification using spatial entanglement

    SciTech Connect

    Sheng Yubo; Deng Fuguo

    2010-10-15

    We present a one-step deterministic entanglement purification protocol with linear optics and postselection. Compared with the Simon-Pan protocol [C. Simon and J. W. Pan, Phys. Rev. Lett. 89, 257901 (2002)], this one-step protocol has some advantages. First, it can obtain a maximally entangled pair from each photon pair with only one step, instead of improving the fidelity of less-entangled photon pairs by performing the entanglement purification process repeatedly in other protocols. Second, it works in a deterministic way, not a probabilistic one, which greatly reduces the number of entanglement resources needed. Third, it does not require the polarization state be entangled; only spatial entanglement is needed. Moreover, it is feasible with current techniques [J. W. Pan, S. Gasparonl, R. Ursin, G. Weihs, and A. Zellinger, Nature (London) 423, 417 (2003)]. All these advantages make this one-step protocol more convenient than others in quantum-communication applications.

  13. Data on single-step purification method for dye-labeled DNA sequencing.

    PubMed

    Fujikura, Kohei

    2016-06-01

    Dye-labelled DNA sequencing is one of the most common and robust technique required for molecular biology since 1977 (Sanger, 1977) [1]. I have recently provided the single-step purification method for dye-labeled sequencing products, which is based on the removal of the washing step in EDTA/ethanol precipitation (Fujikura, 2015) [2]. Here I assess and report the accumulated data of the modified method on the larger scale in practice. PMID:27077088

  14. Single-step purification of native miraculin using immobilized metal-affinity chromatography.

    PubMed

    Duhita, Narendra; Hiwasa-Tanase, Kyoko; Yoshida, Shigeki; Ezura, Hiroshi

    2009-06-24

    Miraculin is a taste-modifying protein that can be isolated from miracle fruit ( Richadella dulcifica ), a shrub native to West Africa. It is able to turn a sour taste into a sweet taste. The commercial exploitation of this sweetness-modifying protein is underway, and a fast and efficient purification method to extract the protein is needed. We succeeded in purifying miraculin from miracle fruit in a single-step purification using immobilized metal-affinity chromatography (IMAC). The purified miraculin exhibited high purity (>95%) in reverse-phase high-performance liquid chromatography. We also demonstrated the necessity of its structure for binding to the nickel-IMAC column. PMID:19469504

  15. Polyethyleneimine assisted-two-step polymerization to develop surface imprinted cryogels for lysozyme purification.

    PubMed

    Erol, Kadir; Köse, Kazım; Uzun, Lokman; Say, Rıdvan; Denizli, Adil

    2016-10-01

    Surface imprinting strategy is one of the promising approaches to synthesize plastic antibodies while overcoming the problems in the protein imprinting research. In this study, we focused our attentions on developing two-step polymerization to imprint on the bare surface employing polyethyleneimine (PEI) assisted-coordination of template molecules, lysozyme. For this aim, we firstly synthesized poly(2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA) cryogels as a bare structure. Then, we immobilized PEI onto the cryogels through the addition reaction between GMA and PEI molecules. After that, we determined the amount of free amine (NH2) groups of PEI molecules, subsequently immobilized methacrylate functionalities onto the half of them and another half was used to chelate Cu(II) ions as a mediator between template, lysozyme and PEI groups. After the characterization of the materials developed by Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and the micro-computed tomography (μCT), we optimized the lysozyme adsorption conditions from aqueous solution. Before performing lysozyme purification from chicken egg white, we evaluated the effects of pH, interaction time, the initial lysozyme concentration, temperature and ionic strength on the lysozyme adsorption. Moreover, the selectivity of surface imprinted cryogels was examined against cytochrome c and bovine serum albumin (BSA) as the competitors. Finally, the mathematical modeling, which was applied to describe the adsorption process, showed that the experimental data is very well-fitted to the Langmuir adsorption isotherm. PMID:27424087

  16. Single-step immunoaffinity purification and characterization of dodecylmaltoside-solubilized human neutrophil flavocytochrome b.

    PubMed

    Taylor, Ross M; Burritt, James B; Foubert, Thomas R; Snodgrass, Meagan A; Stone, Kim C; Baniulis, Danas; Gripentrog, Jeannie M; Lord, Connie; Jesaitis, Algirdas J

    2003-05-01

    Flavocytochrome b (Cyt b) is a heterodimeric, integral membrane protein that serves as the central component of an electron transferase system employed by phagocytes for elimination of bacterial and fungal pathogens. This report describes a rapid and efficient single-step purification of Cyt b from human neutrophil plasma membranes by solubilization in the nonionic detergent dodecylmaltoside (DDM) and immunoaffinity chromatography. A similar procedure for isolation of Cyt b directly from intact neutrophils by a combination of heparin and immunoaffinity chromatography is also presented. The stability of Cyt b was enhanced in DDM relative to previously employed solubilizing agents as determined by both monitoring the heme spectrum in crude membrane extracts and assaying resistance to proteolytic degradation following purification. Gel filtration chromatography and dynamic light scattering indicated that DDM maintains a predominantly monodisperse population of Cyt b following immunoaffinity purification. The high degree of purity obtained with this isolation procedure allowed for direct determination of a 2:1 heme to protein stoichiometry, confirming previous structural models. Analysis of the isolated heterodimer by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry allowed for accurate mass determination of p22(phox) as indicated by the gene sequence. Affinity-purified Cyt b was functionally reconstituted into artificial bilayers and demonstrated that catalytic activity of the protein was efficiently retained throughout the purification procedure. PMID:12729931

  17. Expression and one-step purification of Plasmodium proteins in dictyostelium.

    PubMed

    van Bemmelen, M X; Beghdadi-Rais, C; Desponds, C; Vargas, E; Herrera, S; Reymond, C D; Fasel, N

    2000-12-01

    Nearly full-length Circumsporozoite protein (CSP) from Plasmodium falciparum, the C-terminal fragments from both P. falciparm and P. yoelii CSP and a fragment comprising 351 amino acids of P.vivax MSPI were expressed in the slime mold Dictyostelium discoideum. Discoidin-tag expression vectors allowed both high yields of these proteins and their purification by a nearly single-step procedure. We exploited the galactose binding activity of Discoidin Ia to separate the fusion proteins by affinity chromatography on Sepharose-4B columns. Inclusion of a thrombin recognition site allowed cleavage of the Discoidin-tag from the fusion protein. Partial secretion of the protein was obtained via an ER independent pathway, whereas routing the recombinant proteins to the ER resulted in glycosylation and retention. Yields of proteins ranged from 0.08 to 3 mg l(-1) depending on the protein sequence and the purification conditions. The recognition of purified MSPI by sera from P. vivax malaria patients was used to confirm the native conformation of the protein expressed in Dictyostelium. The simple purification procedure described here, based on Sepharose-4B, should facilitate the expression and the large-scale purification of various Plasmodium polypeptides. PMID:11163444

  18. One-step purification of glucoamylase by affinity precipitation with alginate.

    PubMed

    Teotia, S; Lata, R; Khare, S K; Gupta, M N

    2001-01-01

    It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens. PMID:11746949

  19. A Four-step Approach for Evaluation of Dose Additivity

    EPA Science Inventory

    A four step approach was developed for evaluating toxicity data on a chemical mixture for consistency with dose addition. Following the concepts in the U.S. EPA mixture guidance (EPA 2000), toxicologic interaction for a defined mixture (all components known) is departure from a c...

  20. Purification of equine whole IgG snake antivenom by using an aqueous two phase system as a primary purification step.

    PubMed

    Vargas, Mariángela; Segura, Álvaro; Villalta, Mauren; Herrera, María; Gutiérrez, José María; León, Guillermo

    2015-01-01

    There is a need to introduce innovations in the manufacture of snake antivenoms to increase the supply of these products worldwide. In this work, the fractionation of equine hyperimmune plasma with a new methodology that includes an aqueous two phase system (ATPS) as a primary purification step was compared with the traditional method of caprylic acid precipitation. Hyperimmune plasma from horses immunized with the venoms of three snakes from sub-Saharan Africa was used as starting material for the production of both formulations. After being adjusted to the same lethal neutralizing activity, both antivenoms were compared in terms of their immunoreactivity, neutralization of in vitro venom activities, physicochemical characteristics, and stability. Their performance in terms of yield and purity was also assessed. The neutralization profile of in vitro enzymatic activities and the immunoreactivity, analyzed by ELISA and antivenomic approaches, were very similar for both preparations. Likewise, they behaved similarly in stability studies. However, ATPS-fractionated antivenom showed improved physicochemical profile and immunochemical purity and yield, mainly owing to its lower protein content. Additionally, this methodology allowed the recovery of albumin as a byproduct. ATPS purification constitutes a promising technology for antivenom production and should be further evaluated at preclinical and clinical levels. PMID:25458474

  1. Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization.

    PubMed

    Kober, Daniel L; Yurtsever, Zeynep; Brett, Thomas J

    2015-01-01

    Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case. PMID:26780656

  2. Alternative separation steps for monoclonal antibody purification: combination of centrifugal partitioning chromatography and precipitation.

    PubMed

    Oelmeier, Stefan A; Ladd-Effio, Christopher; Hubbuch, Jürgen

    2013-12-01

    Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography ('CPC') in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein ('HCP'). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process. PMID:24182866

  3. Design of affinity tags for one-step protein purification from immobilized zinc columns

    SciTech Connect

    Pasquinelli, R.S.; Shepherd, R.E.; Koepsel, R.R.; Zhao, A.; Ataai, M.M.

    2000-02-01

    Affinity tags are often used to accomplish recombinant protein purification using immobilized metal affinity chromatography. Success of the tag depends on the chelated metal used and the elution profile of the host cell proteins. Zn(II)-iminodiacetic acid (Zn(II)-IDA) may prove to e superior to either immobilized copper or nickel as a result of its relatively low binding affinity for cellular proteins. for example, almost all Escherichia coli proteins elute from Zn(II)-IDA columns between pH 7.5 and 7.0 with very little cellular protein emerging at pH values lower than 7.0. Thus, a large portion of the Zn(II)-IDA elution profile may be free of contaminant proteins, which can be exploited for one-step purification of a target protein from raw cell extract. In this paper the authors have identified several fusion tags that can direct the elution of the target protein to the low background region of the Zn(II)-IDA elution profile. These tags allow targeting of proteins to different regions of the elution profile, facilitating purification under mild conditions.

  4. 2-step purification of the Ku DNA repair protein expressed in Escherichia coli

    PubMed Central

    2007-01-01

    The Ku protein is involved in DNA double-strand break repair by non-homologous end-joining (NHEJ), which is crucial to the maintenance of genomic integrity in mammals. To study the role of Ku in NHEJ we developed a bicistronic E. coli expression system for the Ku70 and Ku80 subunits. Association of the Ku70 and Ku80 subunits buries a substantial amount of surface area (~9000Å2 [1]), which suggests that herterodimerization may be important for protein stability. N-terminally his6-tagged Ku80 was soluble in the presence, but not in the absence, of bicistronically expressed untagged Ku70. In a 2-step purification, metal chelating affinity chromatography was followed by step-gradient elution from heparin-agarose. Co-purification of equimolar amounts of his6-tagged Ku80 and untagged Ku70 was observed, which indicated heterodimerization. Recombinant Ku bound dsDNA, activated the catalytic subunit of the DNA-dependent kinase (DNA-PKcs) and functioned in NHEJ reactions in vitro. Our results demonstrate that while the heterodimeric interface of Ku is extensive it is nonetheless possible to produce biologically active Ku protein in E. coli. PMID:17110127

  5. Development of an aptamer-affinity chromatography for efficient single step purification of Concanavalin A from Canavalia ensiformis.

    PubMed

    Ahirwar, Rajesh; Nahar, Pradip

    2015-08-01

    Herein, an aptamer-based affinity chromatography method for rapid and single step purification of Concanavalin A is developed and validated. We have used a 41ntssDNA aptamer of Con A (Con A aptabody) as an affinity reagent in the developed aptamer-affinity chromatography. Stationary phase of the method consists of surface functionalized agarose beads carrying covalently immobilized Con A-aptabody. Affinity purification of Con A from jack bean (Canavalia ensiformis) seed using developed aptamer-affinity columns has resulted in ≥66% recovery with 90% purity and 336-fold purification of Con A. The developed aptamer-affinity chromatography has shown efficient scalability and consistent purification when analysed over 13mm, 20mm and 25mm diameter columns having a bed height of 60mm each. Also, the developed aptamer-agarose columns were found to be reusable with recovery decrease of 12.9% in seven sequential cycles of purification. Therefore, the developed aptamer-affinity chromatography provides a novel, efficient and single-step methodology for isolation and purification of Con A. PMID:26102634

  6. One-step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora.

    PubMed

    Krasotkina, Julya; Borisova, Anna A; Gervaziev, Yuri V; Sokolov, Nikolay N

    2004-04-01

    ECAR-LANS, the recombinant L-asparaginase from Erwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K (m) value was 98x10(-6) M for the main physiological substrate L-Asn and 3400x10(-6) M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia. PMID:15032742

  7. One-step chromatographic procedure for purification of B-phycoerythrin from Porphyridium cruentum.

    PubMed

    Tang, Zhihong; Jilu Zhao; Ju, Bao; Li, Wenjun; Wen, Shaohong; Pu, Yang; Qin, Song

    2016-07-01

    B-phycoerythrin (B-PE) was separated and purified from microalga Porphyridium cruentum using one-step chromatographic method. Phycobiliproteins in P. cruentum was extracted by osmotic shock and initially purified by ultrafiltration. Further purification was carried out with a SOURCE 15Q exchange column and analytical grade B-PE was obtained with a purity ratio (A545/A280) of 5.1 and a yield of 68.5%. It showed a double absorption peaks at 545 nm and 565 nm and a shoulder peak at 498 nm, and displayed a fluorescence emission maximum at 580 nm. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a bulky band between 18 and 20 kDa which could be assigned to subunits α and β and a low intensity band of 27 kDa assigned to γ subunit. Our protocol provides attractive alternative to consider for the purification procedure to obtain analytical grade B-PE at commercial level. PMID:26851659

  8. Strategies for the one-step immobilization-purification of enzymes as industrial biocatalysts.

    PubMed

    Barbosa, Oveimar; Ortiz, Claudia; Berenguer-Murcia, Ángel; Torres, Rodrigo; Rodrigues, Rafael C; Fernandez-Lafuente, Roberto

    2015-01-01

    In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize-stabilize-purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review. PMID:25777494

  9. Two-step purification procedure for recombinant human asialoerythropoietin expressed in transgenic plants

    PubMed Central

    Kittur, Farooqahmed S.; Arthur, Elena; Nguyen, Maikhanh; Hung, Chiu-Yueh; Sane, David C.; Xie, Jiahua

    2014-01-01

    Asialoerythropoietin (asialo-EPO) is a desialylated form of human glycoprotein hormone erythropoietin (EPO), which has been reported to be neuro-, cardio-, and renoprotective in animal models of organ injuries. Since the current method of production of asialo-EPO from mammalian cell-made recombinant human EPO (rhuEPOM) by enzymatic desialylation is not commercially viable, we and others used plant-based expression systems to produce recombinant human asialo-EPO (asialo-rhuEPOP). Despite achieving high expression levels in plants, its purification from plant extracts has remained a greater challenge, which has prevented studying its tissue-protective effects and translating it into clinical practice. In this study, a procedure was developed to purify asialo-rhuEPOP from transgenic tobacco leaf tissues in two steps: ion-exchange chromatography based on its high pI (8.75) to separate it from acidic plant proteins, and immunoaffinity chromatography to obtain pure asialo-rhuEPOP. Using this process, up to 31% of the asialo-rhuEPOP could be recovered to near homogeneity from plant extracts. This work demonstrates that asialo-rhuEPOP expressed in tobacco plants could be purified in high yield and purity using minimal steps, which might be suitable for scale-up. Furthermore, the ion-exchange chromatography step together with the use of protein-specific antibody column could be used to purify a wide variety of basic recombinant proteins from transgenic leaf tissues. PMID:25450830

  10. Unusual chromatographic behaviour and one-step purification of a novel membrane proteinase from Bacillus cereus.

    PubMed

    Fricke, B; Buchmann, T; Friebe, S

    1995-11-01

    aggregated at the top of the gel matrix. CCMP solubilized before the purification process, could be eluted in the presence of 0.1% octylphenol-poly(ethyleneglycol ether)9-10 (Triton X-100) in two peaks of Mr 56,000 and 128,000, respectively. We discuss this special chromatographic behaviour of the CCMP from Bacillus cereus, with regard to the strong hydrophobic interactions of the enzyme with the chromatographic matrix and additional self-aggregation, which could only be dissolved by solvents such as isopropanol. PMID:8520670

  11. Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice

    NASA Astrophysics Data System (ADS)

    de Boer, Ernie; Rodriguez, Patrick; Bonte, Edgar; Krijgsveld, Jeroen; Katsantoni, Eleni; Heck, Albert; Grosveld, Frank; Strouboulis, John

    2003-06-01

    Proteomic approaches require simple and efficient protein purification methodologies that are amenable to high throughput. Biotinylation is an attractive approach for protein complex purification due to the very high affinity of avidin/streptavidin for biotinylated templates. Here, we describe an approach for the single-step purification of transcription factor complex(es) based on specific in vivo biotinylation. We expressed the bacterial BirA biotin ligase in mammalian cells and demonstrated very efficient biotinylation of a hematopoietic transcription factor bearing a small (23-aa) artificial peptide tag. Biotinylation of the tagged transcription factor altered neither the factor's protein interactions or DNA binding properties in vivo nor its subnuclear distribution. Using this approach, we isolated the biotin-tagged transcription factor and at least one other known interacting protein from crude nuclear extracts by direct binding to streptavidin beads. Finally, this method works efficiently in transgenic mice, thus raising the prospect of using biotinylation tagging in protein complex purification directly from animal tissues. Therefore, BirA-mediated biotinylation of tagged proteins provides the basis for the single-step purification of proteins from mammalian cells.

  12. One-step purification of R-phycoerythrin from the red edible seaweed Grateloupia turuturu.

    PubMed

    Munier, Mathilde; Morançais, Michèle; Dumay, Justine; Jaouen, Pascal; Fleurence, Joël

    2015-06-15

    A one-step chromatographic method for the purification of R-phycoerythrin (R-PE) of Grateloupia turuturu Yamada is described. Native R-PE was obtained with a purity index of 2.89 and a recovery yield of 27% using DEAE-Sepharose Fast Flow chromatography with a three-step increase in ionic strength. The analysis by SDS electrophoresis showed a broad band between 18 and 21kDa in size corresponding to subunits α and β and a low intensity band of 29kDa corresponding to the γ subunit. Two forms of R-PE were identified by gel filtration chromatography: a native form with a molecular weight of 260±5kDa and a dissociated form with a molecular weight of 60±2kDa. The native form presented the characteristic absorption spectrum of R-PE with three absorbance maxima at 498, 540 and 565nm, whereas the dissociated form presented only the 498 and 540nm peaks. Moreover, the two forms displayed two different fluorescence maxima. PMID:25939094

  13. Semi-Automated Hydrophobic Interaction Chromatography Column Scouting Used in the Two-Step Purification of Recombinant Green Fluorescent Protein

    PubMed Central

    Murphy, Patrick J. M.

    2014-01-01

    Background Hydrophobic interaction chromatography (HIC) most commonly requires experimental determination (i.e., scouting) in order to select an optimal chromatographic medium for purifying a given target protein. Neither a two-step purification of untagged green fluorescent protein (GFP) from crude bacterial lysate using sequential HIC and size exclusion chromatography (SEC), nor HIC column scouting elution profiles of GFP, have been previously reported. Methods and Results Bacterial lysate expressing recombinant GFP was sequentially adsorbed to commercially available HIC columns containing butyl, octyl, and phenyl-based HIC ligands coupled to matrices of varying bead size. The lysate was fractionated using a linear ammonium phosphate salt gradient at constant pH. Collected HIC eluate fractions containing retained GFP were then pooled and further purified using high-resolution preparative SEC. Significant differences in presumptive GFP elution profiles were observed using in-line absorption spectrophotometry (A395) and post-run fluorimetry. SDS-PAGE and western blot demonstrated that fluorometric detection was the more accurate indicator of GFP elution in both HIC and SEC purification steps. Comparison of composite HIC column scouting data indicated that a phenyl ligand coupled to a 34 µm matrix produced the highest degree of target protein capture and separation. Conclusions Conducting two-step protein purification using the preferred HIC medium followed by SEC resulted in a final, concentrated product with >98% protein purity. In-line absorbance spectrophotometry was not as precise of an indicator of GFP elution as post-run fluorimetry. These findings demonstrate the importance of utilizing a combination of detection methods when evaluating purification strategies. GFP is a well-characterized model protein, used heavily in educational settings and by researchers with limited protein purification experience, and the data and strategies presented here may aid in

  14. One-Step Chromatographic Purification of Helicobacter pylori Neutrophil-Activating Protein Expressed in Bacillus subtilis

    PubMed Central

    Shih, Kuo-Shun; Lin, Chih-Chang; Hung, Hsiao-Fang; Yang, Yu-Chi; Wang, Chung-An; Jeng, Kee-Ching; Fu, Hua-Wen

    2013-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP), a major virulence factor of Helicobacter pylori (H. pylori), is capable of activating human neutrophils to produce reactive oxygen species (ROS) and secrete inammatory mediators. HP-NAP is a vaccine candidate, a possible drug target, and a potential in vitro diagnostic marker for H. pylori infection. HP-NAP has also been shown to be a novel therapeutic agent for the treatment of allergic asthma and bladder cancer. Hence, an efficient way to obtain pure HP-NAP needs to be developed. In this study, one-step anion-exchange chromatography in negative mode was applied to purify the recombinant HP-NAP expressed in Bacillus subtilis (B. subtilis). This purification technique was based on the binding of host cell proteins and/or impurities other than HP-NAP to DEAE Sephadex resins. At pH 8.0, almost no other proteins except HP-NAP passed through the DEAE Sephadex column. More than 60% of the total HP-NAP with purity higher than 91% was recovered in the flow-through fraction from this single-step DEAE Sephadex chromatography. The purified recombinant HP-NAP was further demonstrated to be a multimeric protein with a secondary structure of α-helix and capable of activating human neutrophils to stimulate ROS production. Thus, this one-step negative chromatography using DEAE Sephadex resin can efficiently yield functional HP-NAP from B. subtilis in its native form with high purity. HP-NAP purified by this method could be further utilized for the development of new drugs, vaccines, and diagnostics for H. pylori infection. PMID:23577158

  15. Two-step ion-exchange chromatographic purification combined with reversed-phase chromatography to isolate C-peptide for mass spectrometric analysis.

    PubMed

    Kabytaev, Kuanysh; Durairaj, Anita; Shin, Dmitriy; Rohlfing, Curt L; Connolly, Shawn; Little, Randie R; Stoyanov, Alexander V

    2016-02-01

    A liquid chromatography with mass spectrometry on-line platform that includes the orthogonal techniques of ion exchange and reversed phase chromatography is applied for C-peptide analysis. Additional improvement is achieved by the subsequent application of cation- and anion-exchange purification steps that allow for isolating components that have their isoelectric points in a narrow pH range before final reversed-phase mass spectrometry analysis. The utility of this approach for isolating fractions in the desired "pI window" for profiling complex mixtures is discussed. PMID:26717885

  16. Automated harvesting and 2-step purification of unclarified mammalian cell-culture broths containing antibodies.

    PubMed

    Holenstein, Fabian; Eriksson, Christer; Erlandsson, Ioana; Norrman, Nils; Simon, Jill; Danielsson, Åke; Milicov, Adriana; Schindler, Patrick; Schlaeppi, Jean-Marc

    2015-10-30

    Therapeutic monoclonal antibodies represent one of the fastest growing segments in the pharmaceutical market. The growth of the segment has necessitated development of new efficient and cost saving platforms for the preparation and analysis of early candidates for faster and better antibody selection and characterization. We report on a new integrated platform for automated harvesting of whole unclarified cell-culture broths, followed by in-line tandem affinity-capture, pH neutralization and size-exclusion chromatography of recombinant antibodies expressed transiently in mammalian human embryonic kidney 293T-cells at the 1-L scale. The system consists of two bench-top chromatography instruments connected to a central unit with eight disposable filtration devices used for loading and filtering the cell cultures. The staggered parallel multi-step configuration of the system allows unattended processing of eight samples in less than 24h. The system was validated with a random panel of 45 whole-cell culture broths containing recombinant antibodies in the early profiling phase. The results showed that the overall performances of the preparative automated system were higher compared to the conventional downstream process including manual harvesting and purification. The mean recovery of purified material from the culture-broth was 66.7%, representing a 20% increase compared to that of the manual process. Moreover, the automated process reduced by 3-fold the amount of residual aggregates in the purified antibody fractions, indicating that the automated system allows the cost-efficient and timely preparation of antibodies in the 20-200mg range, and covers the requirements for early in vitro and in vivo profiling and formulation of these drug candidates. PMID:26431859

  17. Integrated development of an effective bioprocess for extracellular production of penicillin G acylase in Escherichia coli and its subsequent one-step purification.

    PubMed

    Orr, Valerie; Scharer, Jeno; Moo-Young, Murray; Honeyman, C Howie; Fenner, Drew; Crossley, Lisa; Suen, Shing-Yi; Chou, C Perry

    2012-09-15

    An integrated bioprocess for effective production and purification of penicillin G acylase (PAC) was developed. PAC was overexpressed in a genetically engineered Escherichia coli strain, secreted into the cultivation medium, harvested, and purified in a single step by anion-exchange chromatography. The cultivation medium developed in this study had a sufficiently low conductivity to allow direct application of the extracellular fraction to the anion-exchange chromatography column while providing all of the required nutrients for sustaining cell growth and PAC overexpression. It was contrived with the purposes of (i) providing sufficient osmolarity and buffering capacity, (ii) minimizing ionic species to facilitate the binding of extracellular proteins to anion-exchange media, and (iii) enhancing PAC expression level and secretion efficiency. Employing this medium recipe the specific PAC activity reached a high level at 871 U/g DCW, of which more than 90% was localized in the extracellular medium. In addition, the osmotic pressure and induction conditions were found to be critical for optimal culture performance. The formation of inclusion bodies associated with PAC overexpression tended to arrest cell growth, leading to potential cell lysis. Clarified culture medium was applied to a strong anion-exchange (Q) column and PAC was purified by non-retentive separation, where most contaminant proteins bound to the chromatographic media with PAC being collected as the major component in the flow-through fraction. After removing the contaminant oligopeptides using ultrafiltration, purified PAC with a specific activity of 16.3 U/mg was obtained and the overall purification factor for this one-step downstream purification process was up to 3 fold. PMID:22728392

  18. One step partial purification of beta-D-galactosidase from Kluyveromyces marxianus CDB 002 using STREAMLINE-DEAE.

    PubMed

    Artolozaga, M J; Jonas, R; Schneider, A L; Furlan, S A; Carvalho-Jonas, M de F

    1998-01-01

    The intracellular enzyme beta-D-galactosidase provides interesting applications in the dairy industry, which are able to solve problems related to product processing, or can alleviate lactose intolerance in some populations. In order to obtain a technical enzyme, yeast cells of Kluyveromyces marxianus CDB 002 were disrupted by high pressure homogenization and an innovative chromatographic technique was tested for the recovery of beta-D-galactosidase. A STREAMLINE 25 column, containing 65 ml STREAMLINE-DEAE was equilibrated with 50 mM potassium phosphate buffer pH 7.5 at an upward flow of 250 cmh-1. 100-200 ml cell homogenate were applied onto the expanded gel. After unbound proteins and cellular debris were washed out, the bed was allowed to sediment and beta-D-galactosidase was eluted with a downward flow of 0.2 M NaCl in the same buffer. A 6-fold purification factor was achieved with 63% activity recovery, while removing cell debris at a single step, thus avoiding a centrifugation step. Concentration and volume of the applied sample affected purification and gel performance. The results presented show STREAMLINE-DEAE chromatography to be an interesting method for the production of beta-D-galactosidase as a technical enzyme, since it can also be applied on a large scale without much modification. PMID:10036751

  19. PROLink-Single Step Circularization and Purification Procedure for the Generation of an Improved Variant of Human Growth Hormone.

    PubMed

    Rasche, Nicolas; Tonillo, Jason; Rieker, Marcel; Becker, Stefan; Dorr, Brent; Ter-Ovanesyan, Dmitry; Betz, Ulrich A K; Hock, Björn; Kolmar, Harald

    2016-05-18

    Human growth hormone (hGH) plays an important role during human development and is also an approved therapeutic for the treatment of several diseases. However, one major drawback of hGH is its short circulating half-life requiring frequent administration, which is inconvenient and painful for the patients. Recent publications indicate that circularization greatly increases the stability of proteins due to their protection from exoproteolytic attack and a higher thermal stability of the circular form. Using sortase A, a transpeptidase isolated from Staphylococcus aureus, we developed a single step solid-phase circularization and purification procedure resulting in a circular version of hGH with improved properties. We could show that circular hGH binds to the recombinant hGH receptor with binding kinetics similar to those of linear hGH and that circularization does not alter the biological activity of hGH in vitro. Besides, circular hGH showed almost complete resistance toward exoproteolytic attack and slightly increased thermal stability which could possibly translate into an extended plasma half-life. The single step solid-phase circularization and purification procedure is in principle a generic process, which could also be applied for other proteins that meet the requirements for circularization. PMID:27108993

  20. The Purification of the Chlamydomonas reinhardtii chloroplast ClpP complex: additional subunits and structural features

    PubMed Central

    Derrien, Benoît; Majeran, Wojciech; Effantin, Grégory; Ebenezer, Joseph; Friso, Giulia; van Wijk, Klaas J.; Steven, Alasdair C.; Maurizi, Michael R.; Vallon, Olivier

    2012-01-01

    The ClpP peptidase is a major constituent of the proteolytic machinery of bacteria and organelles. The chloroplast ClpP complex is unusual, in that it associates a large number of subunits, one of which (ClpP1) is encoded in the chloroplast, the others in the nucleus. The complexity of these large hetero-oligomeric complexes has been a major difficulty in their overproduction and biochemical characterization. In this paper, we describe the purification of native chloroplast ClpP complex from the green alga Chlamydomonas reinhardtii, using a strain that carries the Strep-tag II at the C-terminus of the ClpP1 subunit. Similar to land plants, the algal complex comprises active and inactive subunits (3 ClpP and 5 ClpR, respectively). Evidence is presented that a sub-complex can be produced by dissociation, comprising ClpP1 and ClpR1, 2, 3 and 4, similar to the ClpR-ring described in land plants. Our Chlamydomonas ClpP preparation also contains two ClpT subunits, ClpT3 and ClpT4, which like the land plant ClpT1 and ClpT2 show 2 Clp-N domains. ClpTs are believed to function in substrate binding and/or assembly of the two heptameric rings. Phylogenetic analysis indicates that ClpT subunits have appeared independently in Chlorophycean algae, in land plants and in dispersed cyanobacterial genomes. Negative staining electron microscopy shows that the Chlamydomonas complex retains the barrel-like shape of homo-oligomeric ClpPs, with 4 additional peripheral masses that we speculate represent either the additional IS1 domain of ClpP1 (a feature unique to algae) or ClpTs or extensions of ClpR subunits PMID:22772861

  1. Intein-mediated one-step purification of Escherichia coli secreted human antibody fragments.

    SciTech Connect

    Wu, Wan-Yi; Miller, Keith D.; Coolbaugh, Michael; Wood, David W.

    2011-02-25

    In this work, we apply self-cleaving affinity tag technology to several target proteins secreted into the Escherichia coli periplasm, including two with disulfide bonds. The target proteins were genetically fused to a self-cleaving chitin-binding domain intein tag for purification via a chitin agarose affinity resin. By attaching the intein-tagged fusion genes to the PelB secretion leader sequence, the tagged target proteins were secreted to the periplasmic space and could be recovered in active form by simple osmotic shock. After chitin-affinity purification, the target proteins were released from the chitin-binding domain tag via intein self-cleaving. This was induced by a small change in pH from 8.5 to 6.5 at room temperature, allowing direct elution of the cleaved target protein from the chitin affinity resin. The target proteins include the E. coli maltose-binding protein and b-lactamase enzyme, as well as two human antibody fragments that contain disulfide bonds. In all cases, the target proteins were purified with good activity and yield, without the need for refolding. Overall, this work demonstrates the compatibility of the DI-CM intein with the PelB secretion system in E. coli, greatly expanding its potential to more complex proteins.

  2. Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling

    SciTech Connect

    You, C; Zhang, YHP

    2013-02-01

    One-step purification of a multi-enzyme complex was developed based on a mixture of cell extracts containing three dockerin-containing enzymes and one family 3 cellulose-binding module (CBM3)-containing scaffoldin through high-affinity adsorption on low-cost solid regenerated amorphous cellulose (RAC). The three-enzyme complex, called synthetic metabolon, was self-assembled through the high-affinity interaction between the dockerin in each enzyme and three cohesins in the synthetic scaffoldin. The metabolons were either immobilized on the external surface of RAC or free when the scaffoldin contained an intein between the CBM3 and three cohesins. The immobilized and free metabolons containing triosephosphate isomerase, aldolase, and fructose 1,6-biphosphatase exhibited initial reaction rates 48 and 38 times, respectively, that of the non-complexed three-enzyme mixture at the same enzyme loading. Such reaction rate enhancements indicated strong substrate channeling among synthetic metabolons due to the close spatial organization among cascade enzymes. These results suggested that the construction of synthetic metabolons by using cohesins, dockerins, and cellulose-binding modules from cellulosomes not only decreased protein purification labor and cost for in vitro synthetic biology projects but also accelerated reaction rates by 1 order of magnitude compared to non-complexed enzymes. Synthetic metabolons would be an important biocatalytic module for in vitro and in vivo synthetic biology projects.

  3. One-step purification of assembly-competent tubulin from diverse eukaryotic sources

    PubMed Central

    Widlund, Per O.; Podolski, Marija; Reber, Simone; Alper, Joshua; Storch, Marko; Hyman, Anthony A.; Howard, Jonathon; Drechsel, David N.

    2012-01-01

    We have developed a protocol that allows rapid and efficient purification of native, active tubulin from a variety of species and tissue sources by affinity chromatography. The affinity matrix comprises a bacterially expressed, recombinant protein, the TOG1/2 domains from Saccharomyces cerevisiae Stu2, covalently coupled to a Sepharose support. The resin has a high capacity to specifically bind tubulin from clarified crude cell extracts, and, after washing, highly purified tubulin can be eluted under mild conditions. The eluted tubulin is fully functional and can be efficiently assembled into microtubules. The method eliminates the need to use heterologous systems for the study of microtubule-associated proteins and motor proteins, which has been a major issue in microtubule-related research. PMID:22993214

  4. Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus subtilis.

    PubMed

    He, Qing; Fu, Ai-yun; Li, Tian-jiao

    2015-04-01

    The intein expression system has been widely applied in Escherichia coli to express various proteins and peptides. However, the removal of endotoxin from the recombinant proteins expressed in E. coli is very difficult and therefore complicates the purification process. In this study, we constructed an intein-based expression vector for an antimicrobial peptide (cathelicidin from Bungarus fasciatus) and expressed the intein fusion peptide in a Bacillus subtilis expression system. The fusion peptide was secreted into the culture medium, identified by Western blot and purified by affinity chromatography and intein self-cleavage in just one step. Approximately, 0.5 mg peptide was obtained from 1 litre of culture medium. The purified peptide showed antimicrobial activity. Our results indicate that the intein expression system may be a safe and efficient method to produce soluble peptides and proteins in B. subtilis. PMID:25578306

  5. Surface Modified Particles By Multi-Step Addition And Process For The Preparation Thereof

    SciTech Connect

    Cook, Ronald Lee; Elliott, Brian John; Luebben, Silvia DeVito; Myers, Andrew William; Smith, Bryan Matthew

    2006-01-17

    The present invention relates to a new class of surface modified particles and to a multi-step surface modification process for the preparation of the same. The multi-step surface functionalization process involves two or more reactions to produce particles that are compatible with various host systems and/or to provide the particles with particular chemical reactivities. The initial step comprises the attachment of a small organic compound to the surface of the inorganic particle. The subsequent steps attach additional compounds to the previously attached organic compounds through organic linking groups.

  6. One-step purification of functional human and rat pancreatic alpha cells.

    PubMed

    Köhler, Martin; Daré, Elisabetta; Ali, Muhammed Yusuf; Rajasekaran, Subu Surendran; Moede, Tilo; Leibiger, Barbara; Leibiger, Ingo B; Tibell, Annika; Juntti-Berggren, Lisa; Berggren, Per-Olof

    2012-02-01

    Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ~5-fold and ~4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca(2+)](i) upon stimulation with L-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat

  7. Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

    SciTech Connect

    Tykvart, J.; Sacha, P.; Barinka, C.; Knedlik, T.; Starkova, J.; Lubkowski, J.; Konvalinka, J.

    2012-02-07

    Affinity purification is a useful approach for purification of recombinant proteins. Eukaryotic expression systems have become more frequently used at the expense of prokaryotic systems since they afford recombinant eukaryotic proteins with post-translational modifications similar or identical to the native ones. Here, we present a one-step affinity purification set-up suitable for the purification of secreted proteins. The set-up is based on the interaction between biotin and mutated streptavidin. Drosophila Schneider 2 cells are chosen as the expression host, and a biotin acceptor peptide is used as an affinity tag. This tag is biotinylated by Escherichia coli biotin-protein ligase in vivo. We determined that localization of the ligase within the ER led to the most effective in vivo biotinylation of the secreted proteins. We optimized a protocol for large-scale expression and purification of AviTEV-tagged recombinant human glutamate carboxypeptidase II (Avi-GCPII) with milligram yields per liter of culture. We also determined the 3D structure of Avi-GCPII by X-ray crystallography and compared the enzymatic characteristics of the protein to those of its non-tagged variant. These experiments confirmed that AviTEV tag does not affect the biophysical properties of its fused partner. Purification approach, developed here, provides not only a sufficient amount of highly homogenous protein but also specifically and effectively biotinylates a target protein and thus enables its subsequent visualization or immobilization.

  8. Protein ubiquitination via dehydroalanine: development and insights into the diastereoselective 1,4-addition step.

    PubMed

    Meledin, Roman; Mali, Sachitanand M; Singh, Sumeet K; Brik, Ashraf

    2016-06-01

    We report a strategy for site-specific protein ubiquitination using dehydroalanine (Dha) chemistry for the preparation of ubiquitin conjugates bearing a very close mimic of the native isopeptide bond. Our approach relies on the selective formation of Dha followed by conjugation with hexapeptide bearing a thiol handle derived from the C-terminal of ubiquitin. Subsequently, the resulting synthetic intermediate undergoes native chemical ligation with the complementary part of the ubiquitin polypeptide. It has been proposed that the Michael addition step could result in the formation of a diastereomeric mixture as a result of unselective protonation of the enolate intermediate. It has also been proposed that the chiral protein environment may influence such an addition step. In the protein context these questions remain open and no experimental evidence was provided as to how such a protein environment affects the diastereoselectivity of the addition step. As was previously proposed for the conjugation step on protein bearing Dha, the isopeptide bond formation step in our study resulted in the construction of two protein diastereomers. To assign the ratio of these diastereomers, trypsinization coupled with high-pressure liquid chromatography analysis were performed. Moreover, the obtained peptide diastereomers were compared with identical synthetic peptides having defined stereogenic centers, which enabled the determination of the configuration of the isopeptide mimic in each diastereomer. Our study, which offers a new method for isopeptide bond formation and protein ubiquitination, gives insights into the parameters that affect the stereoselectivity of the addition step to Dha for chemical protein modifications. PMID:27143624

  9. One-step Negative Chromatographic Purification of Helicobacter pylori Neutrophil-activating Protein Overexpressed in Escherichia coli in Batch Mode.

    PubMed

    Kuo, Ting-Yu; Hong, Zhi-Wei; Tsai, Chung-Che; Yang, Yu-Chi; Fu, Hua-Wen

    2016-01-01

    Helicobacter pylori neutrophil-activating protein (HP-NAP) is a major virulence factor of Helicobacter pylori (H. pylori). It plays a critical role in H. pylori-induced gastric inflammation by activating several innate leukocytes including neutrophils, monocytes, and mast cells. The immunogenic and immunomodulatory properties of HP-NAP make it a potential diagnostic and vaccine candidate for H. pylori and a new drug candidate for cancer therapy. In order to obtain substantial quantities of purified HP-NAP used for its clinical applications, an efficient method to purify this protein with high yield and purity needs to be established. In this protocol, we have described a method for one-step negative chromatographic purification of recombinant HP-NAP overexpressed in Escherichia coli (E. coli) by using diethylaminoethyl (DEAE) ion-exchange resins (e.g., Sephadex) in batch mode. Recombinant HP-NAP constitutes nearly 70% of the total protein in E. coli and is almost fully recovered in the soluble fraction upon cell lysis at pH 9.0. Under the optimal condition at pH 8.0, the majority of HP-NAP is recovered in the unbound fraction while the endogenous proteins from E. coli are efficiently removed by the resin. This purification method using negative mode batch chromatography with DEAE ion-exchange resins yields functional HP-NAP from E. coli in its native form with high yield and purity. The purified HP-NAP could be further utilized for the prevention, treatment, and prognosis of H. pylori-associated diseases as well as cancer therapy. PMID:27404433

  10. A two-step electrodialysis method for DNA purification from polluted metallic environmental samples.

    PubMed

    Rodríguez-Mejía, José Luis; Martínez-Anaya, Claudia; Folch-Mallol, Jorge Luis; Dantán-González, Edgar

    2008-08-01

    Extracting DNA from samples of polluted environments using standard methods often results in low yields of poor-quality material unsuited to subsequent manipulation and analysis by molecular biological techniques. Here, we report a novel two-step electrodialysis-based method for the extraction of DNA from environmental samples. This technique permits the rapid and efficient isolation of high-quality DNA based on its acidic nature, and without the requirement for phenol-chloroform-isoamyl alcohol cleanup and ethanol precipitation steps. Subsequent PCR, endonuclease restriction, and cloning reactions were successfully performed utilizing DNA obtained by electrodialysis, whereas some or all of these techniques failed using DNA extracted with two alternative methods. We also show that his technique is applicable to purify DNA from a range of polluted and nonpolluted samples. PMID:18601228

  11. Single-step concentration and purification of adenoviruses by coxsackievirus-adenovirus receptor-binding capture and elastin-like polypeptide-mediated precipitation.

    PubMed

    Wu, Qian; Liu, Wenjun; Xu, Bi; Zhang, Xinyu; Xia, Xiaoli; Sun, Huaichang

    2016-02-01

    A single-step method for quick concentration and purification of adenoviruses (Ads) was established by combining coxsackievirus and adenovirus receptor (CAR)-binding capture with elastin-like polypeptide (ELP)-mediated precipitation. The soluble ELP-CAR fusion protein was expressed in vector-transformed E. coli and purified to high purity by two rounds of inverse transition cycling (ITC). After demonstration of the specific binding of fusion protein, a recombinant Ad (rAd), namely rAd/GFP, was pulled down from the culture medium and extract of rAd-transduced cells using ELP-CAR protein, with recovery of 76.2 % and 73.3 %, respectively. The rAd was eluted from the ELP-CAR protein and harvested by one round of ITC, with recoveries ranging from 30.6 % to 34.5 % (virus titration assay). Both ELP-CAR-bound and eluted rAds were able to transduce CAR-positive cells, but not CAR-negative cells (fluorescent microscopy). A further viral titration assay showed that the ELP-CAR-bound rAd/GFP had significantly lower transduction efficiency than the eluted rAd, and there was less of a decrease when tested in the presence of fetal bovine serum. In addition, rAd/GFP was efficiently recovered from the "spiked" PBS and tap water with recovery of ~74 % or ~60 %. This work demonstrates the usefulness of the ELP-CAR-binding capture method for concentration and/or purification of Ads in cellular and environmental samples. PMID:26526147

  12. Immobilized metal ion affinity chromatography on Co2+-carboxymethylaspartate-agarose Superflow, as demonstrated by one-step purification of lactate dehydrogenase from chicken breast muscle.

    PubMed

    Chaga, G; Hopp, J; Nelson, P

    1999-02-01

    A rapid method for the purification of lactate dehydrogenase from whole chicken muscle extract in one chromatographic step is reported. The purification procedure can be accomplished in less than 1 h. A new type of immobilized metal ion affinity chromatography adsorbent is used that can be utilized at linear flow rates higher than 5 cm/min. The final preparation of the enzyme was with purity higher than 95% as ascertained by SDS-PAGE. Three immobilized metal ions (Ni2+, Zn2+ and Co2+) were compared for their binding properties towards the purified enzyme. The binding site of the enzyme for immobilized intermediate metal ions was determined after cleavage with CNBr and binding studies of the derivative peptides on immobilized Co2+. A peptide located on the N-terminus of the enzyme, implicated in the binding, has great potential as a purification tag in fusion proteins. PMID:9889081

  13. Preparative isolation and purification of phlorotannins from Ecklonia cava using centrifugal partition chromatography by one-step.

    PubMed

    Lee, Ji-Hyeok; Ko, Ju-Young; Oh, Jae-Young; Kim, Chul-Young; Lee, Hee-Ju; Kim, Jaeil; Jeon, You-Jin

    2014-09-01

    Various bioactive phlorotannins of Ecklonia cava (e.g., dieckol, eckol, 6,6-bieckol, phloroglucinol, phloroeckol, and phlorofucofuroeckol-A) are reported. However, their isolation and purification are not easy. Centrifugal partition chromatography (CPC) can be used to efficiently purify the various bioactive-compounds efficiently from E. cava. Phlorotannins are successfully isolated from the ethyl acetate (EtOAc) fraction of E. cava by CPC with a two-phase solvent system comprising n-hexane:EtOAc:methanol:water (2:7:3:7, v/v) solution. The dieckol (fraction I, 40.2mg), phlorofucofuroeckol-A (fraction III, 31.1mg), and fraction II (34.1mg) with 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are isolated from the crude extract (500 mg) by a one-step CPC system. The purities of the isolated dieckol and phlorofucofuroeckol-A are ⩾90% according to high performance liquid chromatography (HPLC) and electrospray ionization multi stage tandem mass spectrometry analyses. The purified 2,7-phloroglucinol-6,6-bieckol and pyrogallol-phloroglucinol-6,6-bieckol are collected from fraction II by recycle-HPLC. Thus, the CPC system is useful for easy and simple isolation of phlorotannins from E. cava. PMID:24731366

  14. A Four Step Approach to Evaluate Mixtures for Consistency with Dose Addition

    EPA Science Inventory

    We developed a four step approach for evaluating chemical mixture data for consistency with dose addition for use in environmental health risk assessment. Following the concepts in the U.S. EPA mixture risk guidance (EPA 2000a,b), toxicological interaction for a defined mixture (...

  15. Optimization of isopropanol and ammonium sulfate precipitation steps in the purification of plasmid DNA.

    PubMed

    Freitas, S S; Santos, J A L; Prazeres, D M F

    2006-01-01

    Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 microg pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 microg pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 degrees C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21-23%) without compromising recovery (84-86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (approximately 100%) and purity (32%). In conclusion, it is possible to reduce

  16. Polishing Step Purification of High-Strength Wastewaters by Nanofiltration and Reverse Osmosis.

    PubMed

    Zhou, Jinxiang; Baker, Brian O; Grimsley, Charles T; Husson, Scott M

    2016-01-01

    This article reports findings on the use of nanofiltration (NF) and reverse osmosis (RO) for secondary treatment of high-strength rendering facility wastewaters following an ultrafiltration step. These wastewaters present significant challenges to classical treatment technologies. Constant-pressure, direct-flow membrane filtration experiments were done to screen for flux and effluent water permeate quality of ten commercial NF and RO membranes. All membranes tested were effective in reducing total dissolved salts (TDS) and chemical oxygen demand (COD); however, only two membranes (Koch MPF-34 and Toray 70UB) gave sufficiently stable flux values to warrant longer term cross-flow filtration studies. Cross-flow flux measurements, scanning electron microscopy (SEM), X-ray dispersive spectroscopy (EDS), and attenuated total reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR) indicated that both membranes were eventually fouled by organic and inorganic foulants; however, the Toray 70UB RO membrane yielded a capacity of 1600 L/m² prior to cleaning. A preliminary economic analysis compared the estimated costs of energy and consumables for a dual-stage UF/RO membrane process and dissolved air floatation (DAF) and found membrane process costs could be less than about 40% of the current DAF process. PMID:26978407

  17. Polishing Step Purification of High-Strength Wastewaters by Nanofiltration and Reverse Osmosis

    PubMed Central

    Zhou, Jinxiang; Baker, Brian O.; Grimsley, Charles T.; Husson, Scott M.

    2016-01-01

    This article reports findings on the use of nanofiltration (NF) and reverse osmosis (RO) for secondary treatment of high-strength rendering facility wastewaters following an ultrafiltration step. These wastewaters present significant challenges to classical treatment technologies. Constant-pressure, direct-flow membrane filtration experiments were done to screen for flux and effluent water permeate quality of ten commercial NF and RO membranes. All membranes tested were effective in reducing total dissolved salts (TDS) and chemical oxygen demand (COD); however, only two membranes (Koch MPF-34 and Toray 70UB) gave sufficiently stable flux values to warrant longer term cross-flow filtration studies. Cross-flow flux measurements, scanning electron microscopy (SEM), X-ray dispersive spectroscopy (EDS), and attenuated total reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR) indicated that both membranes were eventually fouled by organic and inorganic foulants; however, the Toray 70UB RO membrane yielded a capacity of 1600 L/m2 prior to cleaning. A preliminary economic analysis compared the estimated costs of energy and consumables for a dual-stage UF/RO membrane process and dissolved air floatation (DAF) and found membrane process costs could be less than about 40% of the current DAF process. PMID:26978407

  18. One-step purification of nucleic acid for gene expression analysis via Immiscible Filtration Assisted by Surface Tension (IFAST)†

    PubMed Central

    Berry, Scott M.; Alarid, Elaine T.; Beebe, David J.

    2011-01-01

    The extraction and purification of nucleic acids from complex samples (e.g. blood, biopsied tissue, cultured cells, food) is an essential prerequisite for many applications in biology including genotyping, transcriptional analysis, systems biology, epigenetic analysis, and virus/bacterial detection. In this report, we describe a new process of nucleic acid extraction that utilizes “pinned” aqueous/organic liquid interfaces in microchannels to streamline the extraction mechanism, replacing all washing steps with a single traverse of an immiscible fluid barrier, termed Immiscible Filtration Assisted by Surface Tension (IFAST). Nucleic acids in biological samples are bound to paramagnetic particles and then drawn across the IFAST device (or array of IFAST devices) using a magnet. While the strength of the IFAST barrier is suitable for separation of nucleic acids from lysate in its current embodiment, its permeability can be selectively adapted by adjusting the surface tensions/energies associated with the cell lysate, the immiscible phase, and the device surface, enabling future expansion to other non-nucleic acid applications. Importantly, processing time is reduced from 15–45 minutes to less than 5 minutes while maintaining purity, yield, and scalability equal to or better than prevailing methods. Operation is extremely simple and no additional lab infrastructure is required. The IFAST technology thus significantly enhances researchers’ abilities to isolate and analyze nucleic acids, a process which is critical and ubiquitous in an extensive array of scientific fields. PMID:21423999

  19. Production and purification of human papillomavirus type 33 L1 virus-like particles from Spodoptera frugiperda 9 cells using two-step column chromatography.

    PubMed

    Baek, Jin-Oh; Seo, Jeong-Woo; Kim, Ik-Hwan; Kim, Chul Ho

    2011-02-01

    The major capsid protein L1 of human papillomavirus (HPV) is essential in construction of recombinant antigen vaccines against cervical cancer. HPV type 33 accounts for about 10% of all HPV infections in Asia. The gene encoding the major capsid protein L1 of the high-risk HPV type 33 was isolated from a Korean patient and expressed in Sf-9 insect cells using a baculovirus expression system. HPV33 L1 protein was isolated by two-step chromatographic purification using strong-cation exchange and ceramic hydroxyapatite chromatography. Strong-cation-exchange chromatography was performed to achieve initial purification of HPV33 L1 and to remove most contaminating proteins, and secondary ceramic hydroxyapatite chromatography yielded pure HPV33 L1 virus-like particles (VLPs). Ceramic hydroxyapatite columns are particularly useful in the purification of antibodies, antigens, human viruses, and VLPs, and we thus used this system. The expression of HPV L1 protein in Sf-9 cells was examined by SDS-PAGE, Western-blotting, and ELISA analyses, and the data showed that HPV33 L1 VLPs were determined to > 98% purity and 58.7% recovery by a quantitative immuno-ELISA assay. Transmission electron microscopy analysis revealed that the HPV VLPs were approximately 50-60 nm in diameter and created by self-assembly of HPV L1 protein. The efficient and simple purification process described here should be useful in production of a cervical cancer vaccine. PMID:20716445

  20. Succinonitrile Purification Facility

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Succinonitrile (SCN) Purification Facility provides succinonitrile and succinonitrile alloys to several NRA selected investigations for flight and ground research at various levels of purity. The purification process employed includes both distillation and zone refining. Once the appropriate purification process is completed, samples are characterized to determine the liquidus and/or solidus temperature, which is then related to sample purity. The lab has various methods for measuring these temperatures with accuracies in the milliKelvin to tenths of milliKelvin range. The ultra-pure SCN produced in our facility is indistinguishable from the standard material provided by NIST to well within the stated +/- 1.5mK of the NIST triple point cells. In addition to delivering material to various investigations, our current activities include process improvement, characterization of impurities and triple point cell design and development. The purification process is being evaluated for each of the four vendors to determine the efficacy of each purification step. We are also collecting samples of the remainder from distillation and zone refining for analysis of the constituent impurities. The large triple point cells developed will contain SCN with a melting point of 58.0642 C +/- 1.5mK for use as a calibration standard for Standard Platinum Resistance Thermometers (SPRTs).

  1. Ribonucleic acid purification.

    PubMed

    Martins, R; Queiroz, J A; Sousa, F

    2014-08-15

    Research on RNA has led to many important biological discoveries and improvement of therapeutic technologies. From basic to applied research, many procedures employ pure and intact RNA molecules; however their isolation and purification are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality. The current techniques to isolate and purify RNA molecules still have several limitations and the requirement for new methods able to improve RNA quality to meet regulatory demands is growing. In fact, as basic research improves the understanding of biological roles of RNAs, the biopharmaceutical industry starts to focus on them as a biotherapeutic tools. Chromatographic bioseparation is a high selective unit operation and is the major option in the purification of biological compounds, requiring high purity degree. In addition, its application in biopharmaceutical manufacturing is well established. This paper discusses the importance and the progress of RNA isolation and purification, considering RNA applicability both in research and clinical fields. In particular and in view of the high specificity, affinity chromatography has been recently applied to RNA purification processes. Accordingly, recent chromatographic investigations based on biorecognition phenomena occurring between RNA and amino acids are focused. Histidine and arginine have been used as amino acid ligands, and their ability to isolate different RNA species demonstrated a multipurpose applicability in molecular biology analysis and RNA therapeutics preparation, highlighting the potential contribution of these methods to overcome the challenges of RNA purification. PMID:24951289

  2. A four-step approach to evaluate mixtures for consistency with dose addition.

    PubMed

    Hertzberg, Richard C; Pan, Yi; Li, Ruosha; Haber, Lynne T; Lyles, Robert H; Herr, David W; Moser, Virginia C; Simmons, Jane Ellen

    2013-11-16

    Mixture risk assessment is often hampered by the lack of dose-response information on the mixture being assessed, forcing reliance on component formulas such as dose addition. We present a four-step approach for evaluating chemical mixture data for consistency with dose addition for use in supporting a component based mixture risk assessment. Following the concepts in the U.S. EPA mixture risk guidance (U.S. EPA, 2000a,b), toxicological interaction for a defined mixture (all components known) is departure from a clearly articulated definition of component additivity. For the common approach of dose additivity, the EPA guidance identifies three desirable characteristics, foremost of which is that the component chemicals are toxicologically similar. The other two characteristics are empirical: the mixture components have toxic potencies that are fixed proportions of each other (throughout the dose range of interest), and the mixture dose term in the dose additive prediction formula, which we call the combined prediction model (CPM), can be represented by a linear combination of the component doses. A consequent property of the proportional toxic potencies is that the component chemicals must share a common dose-response model, where only the dose coefficients depend on the chemical components. A further consequence is that the mixture data must be described by the same mathematical function ("mixture model") as the components, but with a distinct coefficient for the total mixture dose. The mixture response is predicted from the component dose-response curves by using the dose additive CPM and the prediction is then compared with the observed mixture results. The four steps are to evaluate: (1) toxic proportionality by determining how well the CPM matches the single chemical models regarding mean and variance; (2) fit of the mixture model to the mixture data; (3) agreement between the mixture data and the CPM prediction; and (4) consistency between the CPM and the

  3. Fibrous polymer grafted magnetic chitosan beads with strong poly(cation-exchange) groups for single step purification of lysozyme.

    PubMed

    Bayramoglu, Gulay; Tekinay, Turgay; Ozalp, V Cengiz; Arica, M Yakup

    2015-05-15

    Lysozyme is an important polypetide used in medical and food applications. We report a novel magnetic strong cation exchange beads for efficient purification of lysozyme from chicken egg white. Magnetic chitosan (MCHT) beads were synthesized via phase inversion method, and then grafted with poly(glycidyl methacrylate) (p(GMA)) via the surface-initiated atom transfer radical polymerization (SI-ATRP). Epoxy groups of the grafted polymer, were modified into strong cation-exchange groups (i.e., sulfonate groups) in the presence of sodium sulfite. The MCTH and MCTH-g-p(GMA)-SO3H beads were characterized by ATR-FTIR, SEM, and VSM. The sulphonate groups content of the modified MCTH-g-p(GMA)-4 beads was found to be 0.53mmolg(-1) of beads by the potentiometric titration method. The MCTH-g-p(GMA)-SO3H beads were first used as an ion-exchange support for adsorption of lysozyme from aqueous solution. The influence of different experimental parameters such as pH, contact time, and temperature on the adsorption process was evaluated. The maximum adsorption capacity was found to be 208.7mgg(-1) beads. Adsorption of lysozyme on the MCTH-g-p(GMA)-SO3H beads fitted to Langmuir isotherm model and followed the pseudo second-order kinetic. More than 93% of the adsorbed lysozyme was desorbed using Na2CO3 solution (pH 11.0). The purity of the lysozyme was checked by HPLC and SDS gel electrophoresis. In addition, the MCTH-g-p(GMA)-SO3H beads prepared in this work showed promising potential for separation of various anionic molecules. PMID:25864009

  4. One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system.

    PubMed

    Shibusawa, Yoichi; Takeuchi, Naoko; Tsutsumi, Kanako; Nakano, Shigeru; Yanagida, Akio; Shindo, Heisaburo; Ito, Yoichiro

    2007-06-01

    Aqueous-aqueous two-phase (AATP) systems composed of polyethylene glycol (PEG) (molecular mass, M(r):1000-8000) and dextran (M(r):40,000) were evaluated for purification of maltose binding protein tagged-histone deacetylase (MBP-HDAC) by counter-current chromatography (CCC). CCC purification of an MBP-HDAC from Escherichia coli cell-lysate was successfully demonstrated with a 7.0% PEG 3350-10% dextran T40 system containing 10 mM potassium phosphate buffer at pH 9.0. After CCC purification, both polymers in the CCC fractions were easily removed by ultrafiltration in a short period of time. The collected fractions containing target protein were analyzed by an HPLC-based in vitro assay as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis. MBP tag was digested from fusion HDAC during the CCC separation and native HDAC was purified by one-step operation with well preserved deacetyl enzyme activity. PMID:17306809

  5. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner.

    PubMed

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun

    2016-03-01

    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. PMID:26616099

  6. One-step purification of twin-strep-tagged proteins and their complexes on strep-tactin resin cross-linked with bis(sulfosuccinimidyl) suberate (BS3).

    PubMed

    Ivanov, Konstantin I; Bašić, Marta; Varjosalo, Markku; Mäkinen, Kristiina

    2014-01-01

    Affinity purification of Strep-tagged fusion proteins on resins carrying an engineered streptavidin (Strep-Tactin) has become a widely used method for isolation of protein complexes under physiological conditions. Fusion proteins containing two copies of Strep-tag II, designated twin-Strep-tag or SIII-tag, have the advantage of higher affinity for Strep-Tactin compared to those containing only a single Strep-tag, thus allowing more efficient protein purification. However, this advantage is offset by the fact that elution of twin-Strep-tagged proteins with biotin may be incomplete, leading to low protein recovery. The recovery can be dramatically improved by using denaturing elution with sodium dodecyl sulfate (SDS), but this leads to sample contamination with Strep-Tactin released from the resin, making the assay incompatible with downstream proteomic analysis. To overcome this limitation, we have developed a method whereby resin-coupled tetramer of Strep-Tactin is first stabilized by covalent cross-linking with Bis(sulfosuccinimidyl) suberate (BS3) and the resulting cross-linked resin is then used to purify target protein complexes in a single batch purification step. Efficient elution with SDS ensures good protein recovery, while the absence of contaminating Strep-Tactin allows downstream protein analysis by mass spectrometry. As a proof of concept, we describe here a protocol for purification of SIII-tagged viral protein VPg-Pro from nuclei of virus-infected N. benthamiana plants using the Strep-Tactin polymethacrylate resin cross-linked with BS3. The same protocol can be used to purify any twin-Strep-tagged protein of interest and characterize its physiological binding partners. PMID:24796313

  7. Evaluation of Virus and Microbial Purification in Wastewater Soil Absorption Systems Using Multicomponent Surrogate and Tracer Addition

    NASA Astrophysics Data System (ADS)

    Van Cuyk, S.; Siegrist, R. L.

    2001-05-01

    Wastewater soil absorption systems (WSAS) have the potential to achieve high treatment efficiencies, yet the understanding and predictability of performance with respect to removal of viruses and other pathogens remains limited. As part of a long-term program of research to elucidate the fundamental relationships between performance and WSAS process designs and environmental conditions, research has been completed to evaluate virus and microbial purification using multicomponent surrogate and tracer addition. The primary goal of this research was to quantify the removal of virus and bacteria in land-based treatment systems through the use of microbial surrogates and conservative tracers during controlled experiments with 3-D lysimeters in the laboratory and testing of mature WSAS under field conditions. The surrogates and tracers employed to date have included two viruses (MS-2 and PRD-1 bacteriophages), one bacterium (ice-nucleating active (INA) Pseudomonas) and one conservative tracer (bromide). In addition, efforts have been made to determine the relationship between virus and fecal coliforms in soil samples below a WSAS, and the correlation between E.coli concentrations measured in the percolating soil solution as compared to those estimated from analysis of soil solids samples. The results of the research completed to date have revealed that episodic breakthrough of virus and bacteria does occur in WSAS, particularly during early operation, but that a 3-log removal of virus and near complete removal of fecal coliform bacteria can reasonably be expected in WSAS with 60 to 90 cm of sandy medium. Additionally, results from the research indicate that fecal coliforms may be indicative of virus in soil media directly beneath WSAS receiving STE and the concentrations of fecal coliforms in percolating soil solution may be conservatively estimated from analysis of soil solids. Further laboratory and field research is continuing.

  8. Surface Modified Particles By Multi-Step Michael-Type Addition And Process For The Preparation Thereof

    SciTech Connect

    Cook, Ronald Lee; Elliott, Brian John; Luebben, Silvia DeVito; Myers, Andrew William; Smith, Bryan Matthew

    2005-05-03

    A new class of surface modified particles and a multi-step Michael-type addition surface modification process for the preparation of the same is provided. The multi-step Michael-type addition surface modification process involves two or more reactions to compatibilize particles with various host systems and/or to provide the particles with particular chemical reactivities. The initial step comprises the attachment of a small organic compound to the surface of the inorganic particle. The subsequent steps attach additional compounds to the previously attached organic compounds through reactive organic linking groups. Specifically, these reactive groups are activated carbon—carbon pi bonds and carbon and non-carbon nucleophiles that react via Michael or Michael-type additions.

  9. Rapid, two-step purification process for the preparation of pyrogen-free murine immunoglobulin G1 monoclonal antibodies.

    PubMed

    Neidhardt, E A; Luther, M A; Recny, M A

    1992-01-31

    A cost-efficient process was specifically designed for the preparation of gram amounts of highly pure murine immunoglobulin (Ig) G1 monoclonal antibodies (mAbs). This rapid, simple and scalable purification process employs a unique binding and elution protocol for IgG1 mAbs on a silica-based, mixed-mode ion-exchange resin followed by conventional anion-exchange chromatography. mAbs are bound to BakerBond ABx medium at pH 5.6 directly from serum-supplemented hybridoma culture supernatants. Contaminating proteins and nucleic acids are removed by an intermediate wash at pH'6.5, followed by the specific elution of IgG1 mAbs with 100 mM Tris-HCl (pH 8.5). The mAb eluate is then loaded directly on to QAE-Sepharose Fast Flow medium and eluted with 10 mM sodium phosphate buffer (pH 7.4), containing 150 mM sodium chloride. The resulting IgG1 mAbs are greater than 98% pure, free from measurable endotoxin, formulated in a physiological buffer and suitable for in vivo applications. PMID:1560097

  10. Overexpression, one-step purification, and biochemical characterization of a recombinant gamma-glutamyltranspeptidase from Bacillus licheniformis.

    PubMed

    Lin, Long-Liu; Chou, Pei-Ru; Hua, Yu-Wen; Hsu, Wen-Hwei

    2006-11-01

    A truncated gene from Bacillus lichenifromis ATCC 27811 encoding a recombinant gamma-glutamyltranspeptidase (BLrGGT) was cloned into pQE-30 to generate pQE-BLGGT, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-BLGGT) to homogeneity by nickel-chelate chromatography. This protocol yielded over 25 mg of purified BLrGGT per liter of growth culture under optimum conditions. The molecular masses of the subunits of the purified enzyme were determined to be 41 and 22 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum pH and temperature for the recombinant enzyme were 6-8 and 40 degrees C, respectively. The chloride salt of metal ions Mg(2+), K(+), and Na(+) can activate BLrGGT, whereas that of Pb(2+) dramatically inhibited it. The substrate specificity study showed that L-gamma-glutamyl-p-nitroanilide (L-gamma-Glu-p-NA) is a preference for the enzyme. Steady-state kinetic study revealed that BLrGGT has a k (cat) of 105 s(-1) and a K (m) of 21 microM when using L-gamma-Glu-p-NA as the substrate. With this overexpression and purification system, BLrGGT can now be obtained in quantities necessary for structural characterization and synthesis of commercially important gamma-glutamyl compounds. PMID:16850301

  11. A single-step purification and molecular characterization of functional Shiga toxin 2 variants from pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Shiga toxin (Stx) 2 variants, Stx2a, Stx2c, Stx2d and Stx2g were purified to homogeneity from bacterial culture supernatants by a one-step monoclonal anti-Stx affinity chromatography method. The method was based on the binding affinity of these Stxs for a monoclonal antibody against the Stx2 A-subun...

  12. Two step novel hydrogen system using additives to enhance hydrogen release from the hydrolysis of alane and activated aluminum

    SciTech Connect

    Zidan, Ragaiy; Teprovich, Joseph A.; Motyka, Theodore

    2015-12-01

    A system for the generation of hydrogen for use in portable power systems is set forth utilizing a two-step process that involves the thermal decomposition of AlH.sub.3 (10 wt % H.sub.2) followed by the hydrolysis of the activated aluminum (Al*) byproduct to release additional H.sub.2. Additionally, a process in which water is added directly without prior history to the AlH.sub.3:PA composite is also disclosed.

  13. ALUM ADDITION AND STEP-FEED STUDIES IN OXYGEN-ACTIVATED SLUDGE

    EPA Science Inventory

    A plug flow, O2-activated sludge process was operated with alum addition to remove phosphorus and with lime addition to prevent the process pH from decreasing below 6.4. The O2 reactor was operated at F/M ratios between 0.18 to 0.24 gm of BOD5/gm of MLVSS/day in a typical co-curr...

  14. Single step purification of concanavalin A (Con A) and bio-sugar production from jack bean using glucosylated magnetic nano matrix.

    PubMed

    Kim, Ho Myeong; Cho, Eun Jin; Bae, Hyeun-Jong

    2016-08-01

    Jack bean (JB, Canavalia ensiformis) is the source of bio-based products, such as proteins and bio-sugars that contribute to modern molecular biology and biomedical research. In this study, the use of jack bean was evaluated as a source for concanavalin A (Con A) and bio-sugar production. A novel method for purifying Con A from JBs was successfully developed using a glucosylated magnetic nano matrix (GMNM) as a physical support, which facilitated easy separation and purification of Con A. In addition, the enzymatic conversion rate of 2% (w/v) Con A extracted residue to bio-sugar was 98.4%. Therefore, this new approach for the production of Con A and bio-sugar is potentially useful for obtaining bio-based products from jack bean. PMID:26923569

  15. Large-scale single step partial purification of potato pectin methylesterase that enables the use in major food applications.

    PubMed

    Spelbrink, Robin Eric Jacobus; Giuseppin, Marco Luigi Federico

    2014-11-01

    Pectin methylesterase was extracted from potato tubers and partially purified in a single chromatographic step at large industrial scale. The preparation obtained in this way matched the temperature and pH profile of the species reported earlier by Puri et al. (Food Chemistry 8:203-213, 1982) and was enriched 23 times relative to the original potato tubers on a dry matter basis. Potato PME induced gel formation in calcium pectate across a broad pH range and should be suitable for application in the food industry. The procedure presented here represents a sustainable way to recover enzymes from vegetable juices. PMID:25161039

  16. The Effects of Different Modes of Representation on the Solution of One-Step Additive Problems

    ERIC Educational Resources Information Center

    Elia, Iliada; Gagatsis, Athanasios; Demetriou, Andreas

    2007-01-01

    This study investigated the role of different modes of representation, i.e., verbal description, decorative pictures, informational pictures and number line, in solving additive change problems. Data were collected from 1447 students in Grades 1, 2, and 3. Structural equations modelling affirmed the existence of four first-order…

  17. Migration of additive molecules in a polymer filament obtained by melt spinning: Influence of the fiber processing steps

    NASA Astrophysics Data System (ADS)

    Gesta, E.; Skovmand, O.; Espuche, E.; Fulchiron, R.

    2015-12-01

    The purpose of this study is to understand the influence of the yarn processing on the migration of additives molecules, especially insecticide, within polyethylene (PE) yarns. Yarns were manufactured in the laboratory focusing on three key-steps (spinning, post-stretching and heat-setting). Influence of each step on yarn properties was investigated using tensile tests, differential scanning calorimetry and wide-angle X-ray diffraction. The post-stretching step was proved to be critical in defining yarn mechanical and structural properties. Although a first orientation of polyethylene crystals was induced during spinning, the optimal orientation was only reached by post-stretching. The results also showed that the heat-setting did not significantly change these properties. The presence of additives crystals at the yarn surface was evidenced by scanning-electron microscopy. These studies performed at each yarn production step allowed a detailed analysis of the additives' ability to migrate. It is concluded that while post-stretching decreased the migration rate, heat-setting seems to boost this migration.

  18. Preparative two-step purification of recombinant human basic fibroblast growth factor from high-cell-density cultivation of Escherichia coli.

    PubMed

    Garke, G; Deckwer, W D; Anspach, F B

    2000-01-14

    Aggregation and precipitation are major pitfalls during bioprocessing and purification of recombinant human basic fibroblast growth factor (rh-bFGF). In order to gain high yields of the soluble protein monomer with high biological activity, an efficient downstream process was developed, focussing on the combination of expanded bed adsorption (EBA) and heparin chromatography. After expression in E. coli TG1:plambdaFGFB, cells were harvested and washed; then the rh-bFGF was released via high pressure homogenization. The high viscosity of the feedstock of about 40 mPa s, showing non-newtonian behaviour, was reduced to 2 mPa s by the addition of DNase. The homogenate (5.6 l) was loaded directly on an expanded bed column (C-50) packed with the strong cation-exchanger Streamline SP. In the eluates, histone-like (HU) protein was identified as the main protein contaminant by sequence analysis. The thermodynamics and kinetics of rh-bFGF adsorption from the whole broth protein mixture were determined in view of competition and displacement effects with host-derived proteins. Optimal binding and elution conditions were developed with knowledge of the dependence of rh-bFGF adsorption isotherms on the salt concentration to allow direct application of eluates onto Heparin HyperD. This affinity support maintained selectivity and efficiency under CIP and over a wide range of flow-rates; both is advantageous for the flexibility of the purification protocol in view of a scalable process. Remaining DNA and HU protein were separated by Heparin HyperD. The endotoxin level decreased from approximately 1,000,000 EU/ml in the whole broth to 10 EU in 3 mg bFGF per ml. The final purification protocol yields >99% pure rh-bFGF as judged from SDS-PAGE and MALDI-TOF mass spectrometry with high mitogenic activity (ED50=1-1.5 ng/ml) of the lyophilized sample. In comparison to the conventional process, the overall protein recovery rose by 15% to 65% with saving time and costs. PMID:10681038

  19. Expression and single-step purification of mercury transporter (merT) from Cupriavidus metallidurans in E. coli.

    PubMed

    Senthil, K; Gautam, P

    2010-11-01

    The mercury transporter, merT, from Cupriavidus metallidurans was cloned into pRSET-C and expressed in various E. coli hosts. Expression of merT gene failed in common expression hosts like E. coli BL21(DE3), E. coli BL21(DE3)pLysS and E. coli GJ1158 due to expression induced toxicity. The protein was successfully expressed in E. coli C43(DE3) as inclusion bodies. The inclusion bodies were solubilized with Triton X-100 detergent. The detergent solubilized protein with N-terminal His-tag was purified in a single-step by immobilized metal affinity chromatography with a yield of 8 mg l(-1). PMID:20574829

  20. Migration of additive molecules in a polymer filament obtained by melt spinning: Influence of the fiber processing steps

    SciTech Connect

    Gesta, E.; Skovmand, O.; Espuche, E. Fulchiron, R.

    2015-12-17

    The purpose of this study is to understand the influence of the yarn processing on the migration of additives molecules, especially insecticide, within polyethylene (PE) yarns. Yarns were manufactured in the laboratory focusing on three key-steps (spinning, post-stretching and heat-setting). Influence of each step on yarn properties was investigated using tensile tests, differential scanning calorimetry and wide-angle X-ray diffraction. The post-stretching step was proved to be critical in defining yarn mechanical and structural properties. Although a first orientation of polyethylene crystals was induced during spinning, the optimal orientation was only reached by post-stretching. The results also showed that the heat-setting did not significantly change these properties. The presence of additives crystals at the yarn surface was evidenced by scanning-electron microscopy. These studies performed at each yarn production step allowed a detailed analysis of the additives’ ability to migrate. It is concluded that while post-stretching decreased the migration rate, heat-setting seems to boost this migration.

  1. A Single-Step Purification of Cauliflower Lysozyme and Its Dual Role Against Bacterial and Fungal Plant Pathogens.

    PubMed

    Manikandan, Muthu; Balasubramaniam, R; Chun, Se-Chul

    2015-09-01

    A novel lysozyme from cauliflower was purified in a single step, for the first time, using Sephadex G100 column chromatography. The purified lysozyme exhibited a homogenized single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and its molecular mass was calculated to be 22.0 kDa. The purified lysozyme showed activity between 30 to 60 °C with 40 °C as the optimum temperature for its maximal activity. Although the purified lysozyme was functional at pH ranges between 3.0 and 9.0, the optimum pH for the enzyme activity was 8.0. By Michaelis-Menten equation, the threshold substrate concentration for the optimal enzyme activity was calculated to be 133.0 μg. The purified lysozyme showed extraordinary activity against plant pathogenic bacteria and fungi. At 10-μg concentrations, it inhibited the growth of plant pathogenic bacteria such as Pseudomonas syringae, Xanthomonas campestris, and Erwinia carotovora exhibiting 4.28, 5.90, and 3.88-fold inhibition, respectively. Further, it also completely inhibited the conidial germination of Archemonium obclavatum and, to a very large extent, other fungal species such as Fusarium solani (79.3 %), Leptosphaeria maculans (88.6 %), Botrytis cinera (73.3 %), Curvularia lunata (68 %), Rhizoctonia solani (79.6 %), and Alternaria alternata (83.6 %). PMID:26208688

  2. Additives

    NASA Technical Reports Server (NTRS)

    Smalheer, C. V.

    1973-01-01

    The chemistry of lubricant additives is discussed to show what the additives are chemically and what functions they perform in the lubrication of various kinds of equipment. Current theories regarding the mode of action of lubricant additives are presented. The additive groups discussed include the following: (1) detergents and dispersants, (2) corrosion inhibitors, (3) antioxidants, (4) viscosity index improvers, (5) pour point depressants, and (6) antifouling agents.

  3. Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

    PubMed

    Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

    2014-01-15

    Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. PMID:24246428

  4. Engineering foot-and-mouth disease virus serotype O IND R2/1975 for one-step purification by immobilized metal affinity chromatography.

    PubMed

    Biswal, Jitendra K; Bisht, Punam; Subramaniam, Saravanan; Ranjan, Rajeev; Sharma, Gaurav K; Pattnaik, Bramhadev

    2015-09-01

    Immobilized metal affinity chromatography (IMAC) allows for the efficient protein purification via metal affinity tag such as hexa-histidine (His6) sequence. To develop a new chromatography strategy for the purification and concentration of foot-and-mouth disease virus (FMDV) particles, we inserted the His6-tag at the earlier reported site in the VP1 G-H loop of the FMD virus serotype O vaccine strain IND R2/1975. Display of the His6-tag on the capsid surface, endowed the virus with an increased affinity for immobilized nickel ions. We demonstrated that the His6-tagged FMDV could be produced to high titre and purified from the infected BHK-21 cell lysates by IMAC efficiently. Further, a 1150-fold reduction in protein contaminant level and an 8400-fold reduction in DNA contaminant level were achieved in the IMAC purification of His6-tagged FMDV. Through various functional assays it has been found that the tagged virus retains its functionality and infectivity similar to the non-tagged virus. The affinity purification of the His6-tagged FMDV may offer a feasible, alternative approach to the current methods of FMDV antigen purification, concentration and process scalability. PMID:26123433

  5. Process for purification of silicon

    NASA Technical Reports Server (NTRS)

    Rath, H. J.; Sirtl, E.; Pfeiffer, W.

    1981-01-01

    The purification of metallurgically pure silicon having a silicon content of more than 95% by weight is accomplished by leaching with an acidic solution which substantially does not attack silicon. A mechanical treatment leading to continuous particle size reduction of the granulated silicon to be purified is combined with the chemical purification step.

  6. DSPI strain measurement on an externally reinforced bending beam: A comparison of step-by-step addition and pixel shift correlation

    NASA Astrophysics Data System (ADS)

    Hack, Erwin; Schumacher, Ann

    2007-05-01

    A small-scale concrete beam reinforced with an adhesively bonded carbon fiber reinforced polymer (CFRP) plate was subjected to four-point bending. Finite element analyses (FEA) of the bending deformations were carried out to predict strain gradients near the end of the CFRP plate. In order to measure these strains, phase-stepping 3D-digital speckle pattern interferometry was employed. To avoid speckle decorrelation due to the inevitable rigid body motion of the specimen, the load was increased in small increments. Two evaluation schemes for the electronic speckle pattern interferometry phase maps are compared: summing up the measured displacement components load step-by-load step versus regain of the correlation by shifting the final image by an integer number of pixels. Measured strain values are evaluated using a polynomial fit to the measured in-plane displacements and are compared to the FE predicitions. It can be concluded that pixel shift correlation is preferable to summing up load steps for cases of large rigid body motion.

  7. Rapid one-step purification of single-cells encapsulated in alginate microcapsules from oil to aqueous phase using a hydrophobic filter paper: implications for single-cell experiments.

    PubMed

    Lee, Do-Hyun; Jang, Miran; Park, Je-Kyun

    2014-10-01

    By virtue of the biocompatibility and physical properties of hydrogel, picoliter-sized hydrogel microcapsules have been considered to be a biometric signature containing several features similar to that of encapsulated single cells, including phenotype, viability, and intracellular content. To maximize the experimental potential of encapsulating cells in hydrogel microcapsules, a method that enables efficient hydrogel microcapsule purification from oil is necessary. Current methods based on centrifugation for the conventional stepwise rinsing of oil, are slow and laborious and decrease the monodispersity and yield of the recovered hydrogel microcapsules. To remedy these shortcomings we have developed a simple one-step method to purify alginate microcapsules, containing a single live cell, from oil to aqueous phase. This method employs oil impregnation using a commercially available hydrophobic filter paper without multistep centrifugal purification and complicated microchannel networks. The oil-suspended alginate microcapsules encapsulating single cells from mammalian cancer cell lines (MCF-7, HepG2, and U937) and microorganisms (Chlorella vulgaris) were successfully exchanged to cell culture media by quick (~10 min) depletion of the surrounding oil phase without coalescence of neighboring microcapsules. Cell proliferation and high integrity of the microcapsules were also demonstrated by long-term incubation of microcapsules containing a single live cell. We expect that this method for the simple and rapid purification of encapsulated single-cell microcapsules will attain widespread adoption, assisting cell biologists and clinicians in the development of single-cell experiments. PMID:25130499

  8. Isomerization of the uncomplexed actinidin molecule: kinetic accessibility of additional steps in enzyme catalysis provided by solvent perturbation.

    PubMed Central

    Reid, James D; Hussain, Syeed; Bailey, Tamara S F; Sonkaria, Sanjiv; Sreedharan, Suneal K; Thomas, Emrys W; Resmini, Marina; Brocklehurst, Keith

    2004-01-01

    The effects of increasing the content of the aprotic dipolar organic co-solvent acetonitrile on the observed first-order rate constant (k(obs)) of the pre-steady state acylation phases of the hydrolysis of N-acetyl-Phe-Gly methyl thionester catalysed by the cysteine proteinase variants actinidin and papain in sodium acetate buffer, pH 5.3, were investigated by stopped-flow spectral analysis. With low acetonitrile content, plots of k(obs) against [S]0 for the actinidin reaction are linear with an ordinate intercept of magnitude consistent with a five-step mechanism involving a post-acylation conformational change. Increasing the acetonitrile content results in marked deviations of the plots from linearity with a rate minimum around [S]0=150 microM. The unusual negative dependence of k(obs) on [S]0 in the range 25-150 microM is characteristic of a rate-determining isomerization of the free enzyme before substrate binding, additional to the five-step mechanism. There was no evidence for this phenomenon nor for the post-acylation conformational change in the analogous reaction with papain. For this enzyme, however, acetonitrile acts as an inhibitor with approximately uncompetitive characteristics. Possible mechanistic consequences of the differential solvent-perturbed kinetics are indicated. The free enzyme isomerization of actinidin may provide an explanation for the marked difference in sensitivity between this enzyme and papain of binding site-catalytic site signalling in reactions of substrate-derived 2-pyridyl disulphide reactivity probes. PMID:14640975

  9. Adsorption of peptides at the sample drying step: influence of solvent evaporation technique, vial material and solution additive.

    PubMed

    Pezeshki, Adel; Vergote, Valentijn; Van Dorpe, Sylvia; Baert, Bram; Burvenich, Christian; Popkov, Alexander; De Spiegeleer, Bart

    2009-04-01

    Although the efficient and careful removal of solvent from samples by centrifugal evaporation or freeze-drying methods is an important step in peptidomics, the recovery of peptides has not yet been fully investigated with these sample drying methods. Moreover, the surface adsorption of the peptides by the container and efforts to reduce this adsorption by organic additives is only scarcely elaborated until now. In this experiment, the recovery of five model peptides, i.e. bovine insulin, mouse obestatin, goserelin, buserelin and leucine-enkephalin was investigated applying dimethylsulfoxide (DMSO), dimethylformamide (DMF), polyethylene glycol 400 (PEG 400), mannitol and n-nonyl-beta-d-glucopyranoside (C(9)-Glu) in function of the two applied solvent evaporation processes (freeze-drying vs. centrifugal evaporation) and vial types, i.e. polypropylene (PP) and glass. Under our experimental conditions, drying resulted in a decreased recovery of the model peptides by 10% on average. Insulin showed the lowest recovery value relative to the other model peptides. For both drying methods, recovery of the model peptides was increased when C(9)-Glu was present. Overall, the use of PP vials is proposed for freeze-drying, while glass vials are found to be more suitable for centrifugal evaporation. The presence of PEG 400 in PP vials caused significantly reduced recoveries for all model peptides using centrifugal evaporation, although this was not observed in glass vials. As a general conclusion, applying C(9)-Glu as an additive along with choosing appropriate vial type (i.e. PP for lyophilization and glass for centrifugal evaporation) can avoid or diminish peptide loss during the evaporation procedure. PMID:19150589

  10. Hydrogenation of O and OH on Pt(111): A comparison between the reaction rates of the first and the second hydrogen addition steps

    SciTech Connect

    Näslund, L.-Å.

    2014-03-14

    The formation of water through hydrogenation of oxygen on platinum occurs at a surprisingly low reaction rate. The reaction rate limited process for this catalytic reaction is, however, yet to be settled. In the present work, the reaction rates of the first and the second hydrogen addition steps are compared when hydrogen is obtained through intense synchrotron radiation that induces proton production in a water overlayer on top of the adsorbed oxygen species. A substantial amount of the produced hydrogen diffuses to the platinum surface and promotes water formation at the two starting conditions O/Pt(111) and (H{sub 2}O+OH)/Pt(111). The comparison shows no significant difference in the reaction rate between the first and the second hydrogen addition steps, which indicates that the rate determining process of the water formation from oxygen on Pt(111) is neither the first nor the second H addition step or, alternatively, that both H addition steps exert rate control.

  11. Recovery and purification process development for monoclonal antibody production

    PubMed Central

    Ma, Junfen; Winter, Charles; Bayer, Robert

    2010-01-01

    Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

  12. Modeling and robust pooling design of a preparative cation-exchange chromatography step for purification of monoclonal antibody monomer from aggregates.

    PubMed

    Borg, Niklas; Brodsky, Yan; Moscariello, John; Vunnum, Suresh; Vedantham, Ganesh; Westerberg, Karin; Nilsson, Bernt

    2014-09-12

    This study has implemented and calibrated a model that describes the separation of the monomer of monoclonal antibodies from the dimer and larger oligomers on preparative-scale using cation-exchange chromatography. A general rate model with temperature dependent diffusion was coupled to a pH- and temperature-dependent steric mass action model. The model was shown to predict the retention of the monomer, dimer, and oligomer at low loadings for different pH levels and temperatures. Additionally, the model was shown to adequately predict the elution behavior of the monomer and soluble aggregates at high loadings within the same ranges with some limitations. The model was not able to accurately describe the shape of the product break-through curves or the slight levels of co-elution of the dimer and oligomer with the monomer at higher pH. The model was used to predict how 12 process variations impact the separation. The model is used to establish an elution end collection criterion such that the step can robustly provide the target purity of monomers. PMID:25085821

  13. A Four-Step and Four-Criteria Approach for Evaluating Evidence of Dose Addition in Chemical Mixture Toxicity

    EPA Science Inventory

    Dose addition is the most frequently-used component-based approach for predicting dose response for a mixture of toxicologically-similar chemicals and for statistical evaluation of whether the mixture response is consistent with dose additivity and therefore predictable from the ...

  14. Purification process of recombinant monoclonal antibodies with mixed mode chromatography.

    PubMed

    Maria, Sophie; Joucla, Gilles; Garbay, Bertrand; Dieryck, Wilfrid; Lomenech, Anne-Marie; Santarelli, Xavier; Cabanne, Charlotte

    2015-05-01

    An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs. PMID:25805720

  15. Treatment planning of adhesive additive rehabilitations: the progressive wax-up of the three-step technique.

    PubMed

    Vailati, Francesca; Carciofo, Sylvain

    2016-01-01

    A full-mouth rehabilitation should be correctly planned from the start by using a diagnostic wax-up to reduce the potential for remakes, increased chair time, and laboratory costs. However, determining the clinical validity of an extensive wax-up can be complicated for clinicians who lack the experience of full-mouth rehabilitations. The three-step technique is a simplified approach that has been developed to facilitate the clinician's task. By following this technique, the diagnostic wax-up is progressively developed to the final outcome through the interaction between patient, clinician, and laboratory technician. This article provides guidelines aimed at helping clinicians and laboratory technicians to become more proactive in the treatment planning of full-mouth rehabilitations, by starting from the three major parameters of incisal edge position, occlusal plane position, and the vertical dimension of occlusion. PMID:27433550

  16. Catalysis via homolytic substitutions with C-O and Ti-O bonds: oxidative additions and reductive eliminations in single electron steps.

    PubMed

    Gansäuer, Andreas; Fleckhaus, André; Lafont, Manuel Alejandre; Okkel, Andreas; Kotsis, Konstantinos; Anoop, Anakuthil; Neese, Frank

    2009-11-25

    In a combined theoretical and experimental study, an efficient catalytic reaction featuring epoxide opening and tetrahydrofuran formation through homolytic substitution reactions at C-O and Ti-O bonds was devised. The performance of these two key steps of the catalytic cycle was studied and could be adjusted by modifying the electronic properties of the catalysts through introduction of electron-donating or -withdrawing substituents to the titanocene catalysts. By regarding both steps as single electron versions of oxidative addition and reductive elimination, a mechanism-based platform for the design of catalysts and reagents for electron transfer reactions evolved that opens broad perspectives for further investigations. PMID:19919150

  17. Preliminary Hazards Assessment: Iron disulfide purification system

    SciTech Connect

    1991-07-30

    A process for the purification (washing) of iron disulfide (FeS{sub 2}) powder is conducted in the Northeast corner (Area 353) of the main plant building (Building 100). This location is about 130 feet from the fenced boundary of the Partnership School/Child Development Center. In the first steps of the process, raw iron disulfide powder is ground and separated by particle size. The ground and sized powder is then purified in a three-step acid washing process using both hydrochloric acid (HCI) and hydrofluoric (HF) acid. The iron disulfide process is an intermittent batch process conducted four to eight times a year. This study is a Preliminary Hazards Assessment (PHA) to assess the hazards associated with the iron disulfide process. This is a preliminary study and will be used to determine if additional safety analysis is necessary. The scope of the PHA includes assessment of the process steps of grinding, size classification, and purification. The purpose is to identify major hazards and determine if the current and newly added safeguards are adequate for operation. The PHA also lists recommendations for additional safety features that should be added to reduce the risks of operation.

  18. The influence of polymer purification on photovoltaic device performance of a series of indacenodithiophene donor polymers.

    PubMed

    Ashraf, Raja Shahid; Schroeder, Bob C; Bronstein, Hugo A; Huang, Zhenggang; Thomas, Stuart; Kline, R Joseph; Brabec, Christoph J; Rannou, Patrice; Anthopoulos, Thomas D; Durrant, James R; McCulloch, Iain

    2013-04-11

    A series of low bandgap indacenodithiophene polymers is purified by recycling SEC in order to isolate narrow polydispersity fractions. This additional purification step is found to have a significant beneficial influence on the solar cell performance and the reasons for this performance increase are investigated. PMID:23417853

  19. Hamiltonian purification

    SciTech Connect

    Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo; Pascazio, Saverio; Nakazato, Hiromichi; Yuasa, Kazuya; Giovannetti, Vittorio

    2015-12-15

    The problem of Hamiltonian purification introduced by Burgarth et al. [Nat. Commun. 5, 5173 (2014)] is formalized and discussed. Specifically, given a set of non-commuting Hamiltonians (h{sub 1}, …, h{sub m}) operating on a d-dimensional quantum system ℋ{sub d}, the problem consists in identifying a set of commuting Hamiltonians (H{sub 1}, …, H{sub m}) operating on a larger d{sub E}-dimensional system ℋ{sub d{sub E}} which embeds ℋ{sub d} as a proper subspace, such that h{sub j} = PH{sub j}P with P being the projection which allows one to recover ℋ{sub d} from ℋ{sub d{sub E}}. The notions of spanning-set purification and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.

  20. A three-step purification strategy for isolation of hamster TIG2 from CHO cells: characterization of two processed endogenous forms.

    PubMed

    Busmann, Annette; Walden, Michael; Wendland, Martin; Kutzleb, Christian; Forssmann, Wolf-Georg; John, Harald

    2004-11-25

    We have recently isolated a bioactive, circulating protein of human tazarotene-induced gene-2 (TIG2) as the natural ligand of the orphan receptor ChemR23. Here we describe a simplified method for the isolation of hamster TIG2 protein from Chinese hamster ovary (CHO) cell supernatant. Using a heparin-affinity column followed by two reversed phase chromatography steps resulted in the isolation of pure biologically active material. Two processed bioactive forms of Chinese hamster TIG2 were identified by Edman sequencing and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) mass fingerprint analysis, representing the amino acid residues T20 to F156, and T20 to A155 of the 163 amino acid propeptide. Comparison with the predicted aa-sequence indicates a mutation or modification within the C-terminal end of the peptide. PMID:15522723

  1. Direct-immersion solid-phase microextraction coupled to fast gas chromatography mass spectrometry as a purification step for polycyclic aromatic hydrocarbons determination in olive oil.

    PubMed

    Purcaro, Giorgia; Picardo, Massimo; Barp, Laura; Moret, Sabrina; Conte, Lanfranco S

    2013-09-13

    The aim of the present work was to optimize a preparation step for polycyclic aromatic hydrocarbons in a fatty extract. Solid-phase microextraction is an easy preparation technique, which allows to minimize solvent consumption and reduce sample manipulation. A Carbopack Z/polydimethylsiloxane fiber, particularly suitable for extraction of planar compounds, was employed to extract polycyclic aromatic hydrocarbons from a hexane solution obtained after a previous extraction with acetonitrile from oil, followed by a liquid-liquid partition between acetonitrile and hexane. The proposed method was a rapid and sensitive solution to reduce the interference of triglycerides saving the column life and avoiding frequent cleaning of the mass spectrometer ion source. Despite the non-quantitative extraction of polycyclic aromatic hydrocarbons from oil using acetonitrile, the signal-to-noise ratio was significantly improved obtaining a limit of detection largely below the performance criteria required by the European Union legislation. PMID:23915642

  2. Effect of additional etching and ethanol-wet bonding on the dentin bond strength of one-step self-etch adhesives

    PubMed Central

    Ahn, Joonghee; Jung, Kyoung-Hwa; Son, Sung-Ae; Hur, Bock; Kwon, Yong-Hoon

    2015-01-01

    Objectives This study examined the effects of additional acid etching on the dentin bond strength of one-step self-etch adhesives with different compositions and pH. The effect of ethanol wetting on etched dentin bond strength of self-etch adhesives was also evaluated. Materials and Methods Forty-two human permanent molars were classified into 21 groups according to the adhesive types (Clearfil SE Bond [SE, control]; G-aenial Bond [GB]; Xeno V [XV]; Beauti Bond [BB]; Adper Easy Bond [AE]; Single Bond Universal [SU]; All Bond Universal [AU]), and the dentin conditioning methods. Composite resins were placed on the dentin surfaces, and the teeth were sectioned. The microtensile bond strength was measured, and the failure mode of the fractured specimens was examined. The data were analyzed statistically using two-way ANOVA and Duncan's post hoc test. Results In GB, XV and SE (pH ≤ 2), the bond strength was decreased significantly when the dentin was etched (p < 0.05). In BB, AE and SU (pH 2.4 - 2.7), additional etching did not affect the bond strength (p > 0.05). In AU (pH = 3.2), additional etching increased the bond strength significantly (p < 0.05). When adhesives were applied to the acid etched dentin with ethanol-wet bonding, the bond strength was significantly higher than that of the no ethanol-wet bonding groups, and the incidence of cohesive failure was increased. Conclusions The effect of additional acid etching on the dentin bond strength was influenced by the pH of one-step self-etch adhesives. Ethanol wetting on etched dentin could create a stronger bonding performance of one-step self-etch adhesives for acid etched dentin. PMID:25671215

  3. Construction of expression systems for Escherichia coli asparaginase II and two-step purification of the recombinant enzyme from periplasmic extracts.

    PubMed

    Harms, E; Wehner, A; Jennings, M P; Pugh, K J; Beacham, I R; Röhm, K H

    1991-01-01

    Isoenzyme II of Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) is among the few enzymes of major therapeutic importance, being used in the treatment of acute lymphoblastic leukemia. We have constructed several inducible expression systems that overproduce asparaginase II from recombinant plasmids. The most efficient of these systems consists of plasmid pTWE1, a derivative of pT7-7, and an ansB- strain of E. coli, CU1783. These cells produce and secrete amounts of asparaginase II that account for 10-15% of the total cellular protein. Most of the active recombinant enzyme can be released from the periplasmic space by a simple osmotic shock procedure. From the resulting material homogeneous asparaginase II was obtained by a two-step procedure. Overall yields of purified asparaginase were 10-15 mg asparaginase II per liter of E. coli culture. The recombinant enzyme appeared identical to conventionally purified preparations. PMID:1821783

  4. Antibody-Free Magnetic Cell Sorting of Genetically Modified Primary Human CD4+ T Cells by One-Step Streptavidin Affinity Purification

    PubMed Central

    Matheson, Nicholas J.; Peden, Andrew A.; Lehner, Paul J.

    2014-01-01

    Existing methods for phenotypic selection of genetically modified mammalian cells suffer disadvantages of time, cost and scalability and, where antibodies are used to bind exogenous cell surface markers for magnetic selection, typically yield cells coated with antibody-antigen complexes and beads. To overcome these limitations we have developed a method termed Antibody-Free Magnetic Cell Sorting in which the 38 amino acid Streptavidin Binding Peptide (SBP) is displayed at the cell surface by the truncated Low Affinity Nerve Growth Receptor (LNGFRF) and used as an affinity tag for one-step selection with streptavidin-conjugated magnetic beads. Cells are released through competition with the naturally occurring vitamin biotin, free of either beads or antibody-antigen complexes and ready for culture or use in downstream applications. Antibody-Free Magnetic Cell Sorting is a rapid, cost-effective, scalable method of magnetic selection applicable to either viral transduction or transient transfection of cell lines or primary cells. We have optimised the system for enrichment of primary human CD4+ T cells expressing shRNAs and exogenous genes of interest to purities of >99%, and used it to isolate cells following Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 genome editing. PMID:25360777

  5. Expression, purification, and biochemical characterization of recombinant DNA polymerase beta of the Trypanosoma cruzi TcI lineage: requirement of additional factors and detection of phosphorylation of the native form.

    PubMed

    Maldonado, Edio; Rojas, Diego A; Moreira-Ramos, Sandra; Urbina, Fabiola; Miralles, Vicente J; Solari, Aldo; Venegas, Juan

    2015-04-01

    Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major parasitic disease that affects millions of people in America. However, despite the high impact of this disease on human health, no effective and safe treatment has been found that eliminates the infecting parasite from human patients. Among the possible chemotherapeutic targets that could be considered for study in T. cruzi are the DNA polymerases, in particular DNA polymerase beta (polß), which previous studies have shown to be involved in kinetoplast DNA replication and repair. In this paper, we describe the expression, purification, and biochemical characterization of the Miranda clone polß, corresponding to lineage T. cruzi I (TcI). The recombinant enzyme purified to homogeneity displayed specific activity in the range described for a highly purified mammalian polß. However, the trypanosome enzyme exhibited important differences in biochemical properties compared to the mammalian enzymes, specifically an almost absolute dependency on KCl, high sensitivity to N-ethylmaleimide (NEM), and low sensitivity to ddTTP. Immuno-affinity purification of T. cruzi polymerase beta (Tcpolß) from epimastigote extracts showed that the native enzyme was phosphorylated. In addition, it was demonstrated that Tcpolß interacts with some proteins in a group of about 15 proteins which are required to repair 1-6 bases of gaps of a double strand damaged DNA. It is possible that these proteins form part of a DNA repair complex, analogous to that described in mammals and some trypanosomatids. PMID:25566774

  6. [Protein expression and purification].

    PubMed

    Růčková, E; Müller, P; Vojtěšek, B

    2014-01-01

    Production of recombinant proteins is essential for many applications in both basic research and also in medicine, where recombinant proteins are used as pharmaceuticals. This review summarizes procedures involved in recombinant protein expression and purification, including molecular cloning of target genes into expression vectors, selection of the appropriate expression system, and protein purification techniques. Recombinant DNA technology allows protein engineering to modify protein stability, activity and function or to facilitate protein purification by affinity tag fusions. A wide range of cloning systems enabling fast and effective design of expression vectors is currently available. A first choice of protein expression system is usually the bacteria Escherichia coli. The main advantages of this prokaryotic expression system are low cost and simplicity; on the other hand this system is often unsuitable for production of complex mammalian proteins. Protein expression mediated by eukaryotic cells (yeast, insect and mammalian cells) usually produces properly folded and posttranslationally modified proteins. How-ever, cultivation of insect and, especially, mammalian cells is time consuming and expensive. Affinity tagged recombinant proteins are purified efficiently using affinity chromatography. An affinity tag is a protein or peptide that mediates specific binding to a chromatography column, unbound proteins are removed during a washing step and pure protein is subsequently eluted. PMID:24945544

  7. Pool Purification

    NASA Technical Reports Server (NTRS)

    1988-01-01

    Caribbean Clear, Inc. used NASA's silver ion technology as a basis for its automatic pool purifier. System offers alternative approach to conventional purification chemicals. Caribbean Clear's principal markets are swimming pool owners who want to eliminate chlorine and bromine. Purifiers in Caribbean Clear System are same silver ions used in Apollo System to kill bacteria, plus copper ions to kill algae. They produce spa or pool water that exceeds EPA Standards for drinking water.

  8. Polonium purification

    SciTech Connect

    Baker, J.D.

    1996-09-01

    Three processes for the purification of {sup 210}Po from irradiated bismuth targets are described. Safety equipment includes shielded hotcells for the initial separation from other activation products, gloveboxes for handling the volatile and highly toxic materials, and provisions for ventilation. All chemical separations must be performed under vacuum or in inerted systems. Two of the processes require large amounts of electricity; the third requires vessels made from exotic materials.

  9. Single-step purification of Proteus mirabilis urease accessory protein UreE, a protein with a naturally occurring histidine tail, by nickel chelate affinity chromatography.

    PubMed

    Sriwanthana, B; Island, M D; Maneval, D; Mobley, H L

    1994-11-01

    Proteus mirabilis urease, a nickel metalloenzyme, is essential for the virulence of this species in the urinary tract. Escherichia coli containing cloned structural genes ureA, ureB, and ureC and accessory genes ureD, ureE, ureF, and ureG displays urease activity when cultured in M9 minimal medium. To study the involvement of one of these accessory genes in the synthesis of active urease, deletion mutations were constructed. Cultures of a ureE deletion mutant did not produce an active urease in minimal medium. Urease activity, however, was partially restored by the addition of 5 microM NiCl2 to the medium. The predicted amino acid sequence of UreE, which concludes with seven histidine residues among the last eight C-terminal residues (His-His-His-His-Asp-His-His-His), suggested that UreE may act as a Ni2+ chelator for the urease operon. To exploit this potential metal-binding motif, we attempted to purify UreE from cytoplasmic extracts of E. coli containing cloned urease genes. Soluble protein was loaded onto a nickel-nitrilotriacetic acid column, a metal chelate resin with high affinity for polyhistidine tails, and bound protein was eluted with a 0 to 0.5 M imidazole gradient. A single polypeptide of 20-kDa apparent molecular size, as shown by sodium dodecyl sulfate-10 to 20% polyacrylamide gel electrophoresis, was eluted between 0.25 and 0.4 M imidazole. The N-terminal 10 amino acids of the eluted polypeptide exactly matched the deduced amino acid sequence of P. mirabilis UreE. The molecular size of the native protein was estimated on a Superdex 75 column to be 36 kDa, suggesting that the protein is a dimer. These data suggest that UreE is a Ni(2)+-binding protein that is necessary for synthesis of a catalytically active urease at low Ni(2+) concentrations. PMID:7961442

  10. PURIFICATION PROCESS

    DOEpatents

    Wibbles, H.L.; Miller, E.I.

    1958-01-14

    This patent deals with the separation of uranium from molybdenum compounds, and in particular with their separation from ether solutions containing the molybdenum in the form of acids, such as silicomolybdic and phosphomolybdic acids. After the nitric acid leach of pitchblende, the molybdenum values present in the ore are found in the leach solution in the form of complex acids. The uranium bearing solution may be purified of this molybdenum content by comtacting it with activated charcoal. The purification is improved when the acidity of the solution is low ad agitation is also beneficial. The molybdenum may subsequently be recovered from the charcosl ad the charcoal reused.

  11. Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    Silver ionization water purification technology was originally developed for Apollo spacecraft. It was later used to cleanse swimming pools and has now been applied to industrial cooling towers and process coolers. Sensible Technologies, Inc. has added two other technologies to the system, which occupies only six square feet. It is manufactured in three capacities, and larger models are custom built on request. The system eliminates scale, corrosion, algae, bacteria and debris, and because of the NASA technology, viruses and waterborne bacteria are also destroyed. Applications include a General Motors cooling tower, amusement parks, ice manufacture and a closed-loop process cooling system.

  12. Optimized Expression and Purification for High-Activity Preparations of Algal [FeFe]-Hydrogenase

    SciTech Connect

    Yacoby, I.; Tegler, L. T.; Pochekailov, S.; Zhang, S.; King, P. W.

    2012-04-01

    Recombinant expression and purification of metallo-enzymes, including hydrogenases, at high-yields is challenging due to complex, and enzyme specific, post-translational maturation processes. Low fidelities of maturation result in preparations containing a significant fraction of inactive, apo-protein that are not suitable for biophysical or crystallographic studies. We describe the construction, overexpression and high-yield purification of a fusion protein consisting of the algal [2Fe2S]-ferredoxin PetF (Fd) and [FeFe]-hydrogenase HydA1. The maturation of Fd-HydA1 was optimized through improvements in culture conditions and media components used for expression. We also demonstrated that fusion of Fd to the N-terminus of HydA1, in comparison to the C-terminus, led to increased expression levels that were 4-fold higher. Together, these improvements led to enhanced HydA1 activity and improved yield after purification. The strong binding-affinity of Fd for DEAE allowed for two-step purification by ion exchange and StrepTactin affinity chromatography. In addition, the incorporation of a TEV protease site in the Fd-HydA1 linker allowed for the proteolytic removal of Fd after DEAE step, and purification of HydA1 alone by StrepTactin. In combination, this process resulted in HydA1 purification yields of 5 mg L{sup -1} of culture from E. coli with specific activities of 1000 U (U = 1 {micro}mol hydrogen evolved mg{sup -1} min{sup -1}). The [FeFe]-hydrogenases are highly efficient enzymes and their catalytic sites provide model structures for synthetic efforts to develop robust hydrogen activation catalysts. In order to characterize their structure-function properties in greater detail, and to use hydrogenases for biotechnological applications, reliable methods for rapid, high-yield expression and purification are required.

  13. Semiconductor grade, solar silicon purification project

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Rosler, R. R.; Thompson, S. W.; Chaney, R. E.

    1979-01-01

    Experimental apparatus and procedures used in the development of a 3-step SiF2(x) polymer transport purification process are described. Both S.S.M.S. and E.S. analysis demonstrated that major purification had occured and some samples were indistinguishable from semiconductor grade silicon (except possibly for phosphorus). Recent electrical analysis via crystal growth reveals that the product contains compensated phosphorus and boron. The low projected product cost and short energy payback time suggest that the economics of this process will result in a cost less than the goal of $10/Kg(1975 dollars). The process appears to be readily scalable to a major silicon purification facility.

  14. Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems.

    PubMed

    Li, Mian; Peeples, Tonya L

    2004-07-25

    Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes. PMID:15177162

  15. High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

    PubMed Central

    Luo, Dan; Wen, Caixia; Zhao, Rongchuan; Liu, Xinyu; Liu, Xinxin; Cui, Jingjing; Liang, Joshua G.; Liang, Peng

    2016-01-01

    Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine—5’) pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake. PMID:27214237

  16. Sodium Benzoate, a Food Additive and a Metabolite of Cinnamon, Modifies T Cells at Multiple Steps and Inhibits Adoptive Transfer of Experimental Allergic Encephalomyelitis1

    PubMed Central

    Brahmachari, Saurav; Pahan, Kalipada

    2007-01-01

    Experimental allergic encephalomyelitis (EAE) is the animal model for multiple sclerosis. This study explores a novel use of sodium benzoate (NaB), a commonly used food additive and a Food and Drug Administration-approved nontoxic drug for urea cycle disorders, in treating the disease process of relapsing-remitting EAE in female SJL/J mice. NaB, administered through drinking water at physiologically tolerable doses, ameliorated clinical symptoms and disease progression of EAE in recipient mice and suppressed the generation of encephalitogenic T cells in donor mice. Histological studies reveal that NaB effectively inhibited infiltration of mononuclear cells and demyelination in the spinal cord of EAE mice. Consequently, NaB also suppressed the expression of proinflammatory molecules and normalized myelin gene expression in the CNS of EAE mice. Furthermore, we observed that NaB switched the differentiation of myelin basic protein-primed T cells from Th1 to Th2 mode, enriched regulatory T cell population, and down-regulated the expression of various contact molecules in T cells. Taken together, our results suggest that NaB modifies encephalitogenic T cells at multiple steps and that NaB may have therapeutic importance in multiple sclerosis. PMID:17579047

  17. Evaluation of corn oil as an additive in the pre-enrichment step to increase recovery of Salmonella enterica from oregano.

    PubMed

    Jean-Gilles Beaubrun, Junia; Flamer, Marie-Laure; Addy, Nicole; Ewing, Laura; Gopinath, Gopal; Jarvis, Karen; Grim, Chris; Hanes, Darcy E

    2016-08-01

    Phenolic compounds associated with essential oils of spices and herbs possess a variety of antioxidant and antimicrobial properties that interfere with Salmonella detection from fresh and dried products. Finding a compound to neutralize the effect of these antimicrobial compounds, while allowing Salmonella growth during pre-enrichment, is a crucial step in both traditional pathogen isolation and molecular detection from these foods. This study evaluated the effectiveness of corn oil as a component of the pre-enrichment broth to counteract antimicrobial compounds properties and increase the recovery of Salmonella from spices. Oregano samples artificially contaminated with Salmonella enterica were pre-enriched in modified Buffered Peptone Water (mBPW) supplemented with and without 2% (vol/vol) corn oil respectively. Samples were incubated overnight at 37 °C. The results showed that recovery of Salmonella from oregano samples was increased by ≥50% when pre-enriched with corn oil. Serovars were confirmed using a PCR serotyping method. In addition, shot-gun metagenomics analyses demonstrated bacterial diversity and the effect of corn oil on the relative prevalence of Salmonella in the oregano samples. Modifying pre-enrichment broths with corn oil improved the detection and isolation of Salmonella from oregano, and may provide an alternative method for pathogen detection in dried food matrices such as spices. PMID:27052719

  18. Traceless purification and desulfurization of tau protein ligation products.

    PubMed

    Reimann, Oliver; Smet-Nocca, Caroline; Hackenberger, Christian P R

    2015-01-01

    We present a novel strategy for the traceless purification and synthetic modification of peptides and proteins obtained by native chemical ligation. The strategy involves immobilization of a photocleavable semisynthetic biotin-protein conjugate on streptavidin-coated agarose beads, which eliminates the need for tedious rebuffering steps and allows the rapid removal of excess peptides and additives. On-bead desulfurization is followed by delivery of the final tag-free protein product. The strategy is demonstrated in the isolation of a tag-free Alzheimer's disease related human tau protein from a complex EPL mixture as well as a triphosphorylated peptide derived from the C-terminus of tau. PMID:25404175

  19. Next Step for STEP

    SciTech Connect

    Wood, Claire; Bremner, Brenda

    2013-08-09

    The Siletz Tribal Energy Program (STEP), housed in the Tribe’s Planning Department, will hire a data entry coordinator to collect, enter, analyze and store all the current and future energy efficiency and renewable energy data pertaining to administrative structures the tribe owns and operates and for homes in which tribal members live. The proposed data entry coordinator will conduct an energy options analysis in collaboration with the rest of the Siletz Tribal Energy Program and Planning Department staff. An energy options analysis will result in a thorough understanding of tribal energy resources and consumption, if energy efficiency and conservation measures being implemented are having the desired effect, analysis of tribal energy loads (current and future energy consumption), and evaluation of local and commercial energy supply options. A literature search will also be conducted. In order to educate additional tribal members about renewable energy, we will send four tribal members to be trained to install and maintain solar panels, solar hot water heaters, wind turbines and/or micro-hydro.

  20. Rapid Microscale Isolation and Purification of Yeast Alcohol Dehydrogenase Using Cibacron Blue Affinity Chromatography

    NASA Astrophysics Data System (ADS)

    Morgan, Chad; Moir, Neil

    1996-11-01

    A rapid microscale procedure has been developed for the isolation and purification of yeast alcohol dehydrogenase. Glass beads are used for cytolysis, PEG precipitation for partial purification and a cibacron blue affinity column for the final step. A 27.5 fold purification can be achieved in 2 - 3 hours.

  1. Exhaust gas purification device

    SciTech Connect

    Fujiwara, H.; Hibi, T.; Sayo, S.; Sugiura, Y.; Ueda, K.

    1980-02-19

    The exhaust gas purification device includes an exhaust manifold , a purification cylinder connected with the exhaust manifold through a first honey-comb shaped catalyst, and a second honeycomb shaped catalyst positioned at the rear portion of the purification cylinder. Each catalyst is supported by steel wool rings including coarse and dense portions of steel wool. The purification device further includes a secondary air supplying arrangement.

  2. Affinity purification of heme-tagged proteins.

    PubMed

    Asher, Wesley B; Bren, Kara L

    2014-01-01

    Protein affinity purification techniques are widely used for isolating pure target proteins for biochemical and structural characterization. Herein, we describe the protocol for affinity-based purification of proteins expressed in Escherichia coli that uses the coordination of a peptide tag covalently modified with heme c, known as a heme-tag, to an L-histidine immobilized Sepharose resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. In addition, we describe methods for specifically detecting heme-tagged proteins in SDS-PAGE gels using a heme-staining procedure and for quantifying the proteins using a pyridine hemochrome assay. PMID:24943311

  3. One-step synthesis of quinazolino[3,2-a]quinazolinones via palladium-catalyzed domino addition/carboxamidation reactions.

    PubMed

    Zeng, Fanlong; Alper, Howard

    2010-08-20

    A highly efficient palladium-catalyzed domino process has been developed for the synthesis of quinazolino[3,2-a]quinazolinones by forming five new bonds in a single step. Despite the high density and variety of functional groups on the substrates, the tetracyclic quinazolinones were obtained in good to excellent yields. PMID:20666363

  4. Economic Methods of Ginger Protease'sextraction and Purification

    NASA Astrophysics Data System (ADS)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  5. Strep-Tagged Protein Purification.

    PubMed

    Maertens, Barbara; Spriestersbach, Anne; Kubicek, Jan; Schäfer, Frank

    2015-01-01

    The Strep-tag system can be used to purify recombinant proteins from any expression system. Here, protocols for lysis and affinity purification of Strep-tagged proteins from E. coli, baculovirus-infected insect cells, and transfected mammalian cells are given. Depending on the amount of Strep-tagged protein in the lysate, a protocol for batch binding and subsequent washing and eluting by gravity flow can be used. Agarose-based matrices with the coupled Strep-Tactin ligand are the resins of choice, with a binding capacity of up to 9 mg ml(-1). For purification of lower amounts of Strep-tagged proteins, the use of Strep-Tactin magnetic beads is suitable. In addition, Strep-tagged protein purification can also be automated using prepacked columns for FPLC or other liquid-handling chromatography instrumentation, but automated purification is not discussed in this protocol. The protocols described here can be regarded as an update of the Strep-Tag Protein Handbook (Qiagen, 2009). PMID:26096503

  6. Security: Step by Step

    ERIC Educational Resources Information Center

    Svetcov, Eric

    2005-01-01

    This article provides a list of the essential steps to keeping a school's or district's network safe and sound. It describes how to establish a security architecture and approach that will continually evolve as the threat environment changes over time. The article discusses the methodology for implementing this approach and then discusses the…

  7. Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach.

    PubMed Central

    Burnet, M.; Lafontaine, P. J.; Hanson, A. D.

    1995-01-01

    The osmoprotectant glycine betaine is synthesized via the path-way choline -> betaine aldehyde -> glycine betaine. In spinach (Spinacia oleracea), the first step is catalyzed by choline monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO. [14C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [14C]betaine aldehyde to [14C]glycine betaine, which is isolated by ion exchange. The assay was used in the purification of CMO from leaves of salinized spinach. The 10-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about 600-fold and recovery of activity was 0.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a polypeptide with a molecular mass of 45 kD. Taken with the value of 98 kD estimated for native CMO (R. Brouquisse, P. Weigel, D. Rhodes, C.F. Yocum, A.D. Hanson [1989] Plant Physiol 90: 322-329), this indicates that CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein. PMID:12228495

  8. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase.

    PubMed

    Jaffee, Marcie B; Imperiali, Barbara

    2013-06-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of Escherichia coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  9. Optimized protocol for expression and purification of membrane-bound PglB, a bacterial oligosaccharyl transferase

    PubMed Central

    Jaffee, Marcie B.; Imperiali, Barbara

    2013-01-01

    Asparagine-linked glycosylation (NLG) plays a significant role in a diverse range of cellular processes, including protein signaling and trafficking, the immunologic response, and immune system evasion by pathogens. A major impediment to NLG-related research is an incomplete understanding of the central enzyme in the biosynthetic pathway, the oligosaccharyl transferase (OTase). Characterization of the OTase is critical for developing ways to inhibit, engineer, and otherwise manipulate the enzyme for research and therapeutic purposes. The minimal understanding of this enzyme can be attributed to its complex, transmembrane structure, and the resulting instability and resistance to overexpression and purification. The following article describes an optimized procedure for recombinant expression and purification of PglB, a bacterial OTase, in a stably active form. The conditions screened at each step, the order of screening, and the method of comparing conditions are described. Ultimately, the following approach increased expression levels from tens of micrograms to several milligrams of active protein per liter of E. coli culture, and increased stability from several hours to greater than six months post-purification. This represents the first detailed procedure for attaining a pure, active, and stable OTase in milligram quantities. In addition to presenting an optimized protocol for expression and purification of PglB, these results present a general guide for the systematic optimization of the expression, purification, and stability of a large, transmembrane protein. PMID:23583934

  10. Towards a single step process to create high purity gold structures by electron beam induced deposition at room temperature.

    PubMed

    Mansilla, C; Mehendale, S; Mulders, J J L; Trompenaars, P H F

    2016-10-14

    Highly pure metallic structures can be deposited by electron beam induced deposition and they have many important applications in different fields. The organo-metallic precursor is decomposed and deposited under the electron beam, and typically it is purified with post-irradiation in presence of O2. However, this approach limits the purification to the surface of the deposit. Therefore, 'in situ' purification during deposition using simultaneous flows of both O2 and precursor in parallel with two gas injector needles has been tested and verified. To simplify the practical arrangements, a special concentric nozzle has been designed allowing deposition and purification performed together in a single step. With this new device metallic structures with high purity can be obtained more easily, while there is no limit on the height of the structures within a practical time frame. In this work, we summarize the first results obtained for 'in situ' Au purification using this concentric nozzle, which is described in more detail, including flow simulations. The operational parameter space is explored in order to optimize the shape as well as the purity of the deposits, which are evaluated through scanning electron microscope and energy dispersive x-ray spectroscopy measurements, respectively. The observed variations are interpreted in relation to other variables, such as the deposition yield. The resistivity of purified lines is also measured, and the influence of additional post treatments as a last purification step is studied. PMID:27587078

  11. Optimized entanglement purification schemes for modular based quantum computers

    NASA Astrophysics Data System (ADS)

    Krastanov, Stefan; Jiang, Liang

    The choice of entanglement purification scheme strongly depends on the fidelities of quantum gates and measurements, as well as the imperfection of initial entanglement. For instance, the purification scheme optimal at low gate fidelities may not necessarily be the optimal scheme at higher gate fidelities. We employ an evolutionary algorithm that efficiently optimizes the entanglement purification circuit for given system parameters. Such optimized purification schemes will boost the performance of entanglement purification, and consequently enhance the fidelity of teleportation-based non-local coupling gates, which is an indispensible building block for modular-based quantum computers. In addition, we study how these optimized purification schemes affect the resource overhead caused by error correction in modular based quantum computers.

  12. Purification of Carbon Nanotubes: Alternative Methods

    NASA Technical Reports Server (NTRS)

    Files, Bradley; Scott, Carl; Gorelik, Olga; Nikolaev, Pasha; Hulse, Lou; Arepalli, Sivaram

    2000-01-01

    Traditional carbon nanotube purification process involves nitric acid refluxing and cross flow filtration using surfactant TritonX. This is believed to result in damage to nanotubes and surfactant residue on nanotube surface. Alternative purification procedures involving solvent extraction, thermal zone refining and nitric acid refiuxing are used in the current study. The effect of duration and type of solvent to dissolve impurities including fullerenes and P ACs (polyaromatic compounds) are monitored by nuclear magnetic reasonance, high performance liquid chromatography, and thermogravimetric analysis. Thermal zone refining yielded sample areas rich in nanotubes as seen by scanning electric microscopy. Refluxing in boiling nitric acid seem to improve the nanotube content. Different procedural steps are needed to purify samples produced by laser process compared to arc process. These alternative methods of nanotube purification will be presented along with results from supporting analytical techniques.

  13. Purification process for vertically aligned carbon nanofibers

    NASA Technical Reports Server (NTRS)

    Nguyen, Cattien V.; Delziet, Lance; Matthews, Kristopher; Chen, Bin; Meyyappan, M.

    2003-01-01

    Individual, free-standing, vertically aligned multiwall carbon nanotubes or nanofibers are ideal for sensor and electrode applications. Our plasma-enhanced chemical vapor deposition techniques for producing free-standing and vertically aligned carbon nanofibers use catalyst particles at the tip of the fiber. Here we present a simple purification process for the removal of iron catalyst particles at the tip of vertically aligned carbon nanofibers derived by plasma-enhanced chemical vapor deposition. The first step involves thermal oxidation in air, at temperatures of 200-400 degrees C, resulting in the physical swelling of the iron particles from the formation of iron oxide. Subsequently, the complete removal of the iron oxide particles is achieved with diluted acid (12% HCl). The purification process appears to be very efficient at removing all of the iron catalyst particles. Electron microscopy images and Raman spectroscopy data indicate that the purification process does not damage the graphitic structure of the nanotubes.

  14. Expression and purification of GST fusion proteins.

    PubMed

    Harper, S; Speicher, D W

    2001-05-01

    An increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a "tag" that provides the basis for rapid high-affinity purification. This unit describes the expression and purification of fusion proteins containing the 26-kDa glutathione-S-transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high-affinity one-step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag. PMID:18429193

  15. Efficient lipase purification using reverse micellar extraction.

    PubMed

    Gaikaiwari, Raghavendra P; Wagh, Shilpa A; Kulkarni, Bhaskar D

    2012-03-01

    Reverse micellar extraction (RME) of enzyme provides an attractive option for conventional method with the potential to achieve purification and concentration in a single step with high yield. This study presents a methodology for optimization of RME with Pseudomonas lipase as model system. Fold-purification, percent recovery and extraction time were the objective functions while the type and concentration of surfactant, contact time, pH, ionic strength, and the ratio of organic to aqueous phase were the decision variables. Under optimized conditions, the AOT (Aerosol OT (bis 2-ethylhexyl) sodium sulfosuccinate)-isooctane system gave a 15-fold purification, 80% recovery and 2.5-fold concentration of the Pseudomonas lipase with process time of 45 min. PMID:22230773

  16. Purification of DNA Oligos by denaturing polyacrylamide gel electrophoresis (PAGE).

    PubMed

    Lopez-Gomollon, Sara; Nicolas, Francisco Esteban

    2013-01-01

    After chemical synthesis, the oligonucleotide preparation contains the desired full-length oligonucleotide but also all of the DNA molecules that were aborted during each cycle in the synthesis, and the by-products generated during the chemical reactions. The purification of oligonucleotides is a critical step for demanding applications where the exact length or sequence of the oligonucleotide is important, or for oligonucleotides longer than 50 bases. There are several methods of increasing oligonucleotide purity, the choice of which will depend on modifications of the oligonucleotides and their intended use. Polyacrylamide gel purification (PAGE purification) is the method of choice when the highest percentage of full-length oligonucleotide is desired. This chapter describes a protocol for oligonucleotide purification using denaturing polyacrylamide gel electrophoresis, and includes oligonucleotide preparation, polyacrylamide gel electrophoresis, and purification from the gel slice by two different methods: by diffusion or by electroelution. This chapter also includes recommendations as well as protocol advice. PMID:24011037

  17. Strategies and considerations for protein purifications.

    PubMed

    Linn, Stuart

    2009-01-01

    Prior to embarking upon the purification of a protein, one should begin by considering what the protein is to be used for. In particular, how much of the protein is needed, what should be its state of purity, and must it be folded correctly and associated with various other peptides or cofactors. Using such criteria, an appropriate assay should be chosen and a procedure be planned taking into account the source of the protein, how it is to be extracted from the source, and what agents the protein ought to be exposed to or ultimately be stored in. One is often surprised at the time necessary to develop an appropriate protein purification procedure relative to the time required to clone a gene or to accumulate information with the purified protein. There are an overwhelming number of options for protein purification steps, so forethought is necessary to expedite the tedious job of developing the purification scheme, or to avoid having to redesign it upon attempting to use the protein. This chapter points out general considerations to be undertaken in designing, organizing, and executing the purification, while subsequent chapters of this volume supply more specific options and technical details. PMID:19892162

  18. Optimization of purification method and characterization of recombinant human Centrin-1.

    PubMed

    Phanindranath, Regur; Sudhakar, Digumarthi V S; Sharma, Anand Kumar; Thangaraj, Kumarasamy; Sharma, Yogendra

    2016-08-01

    Centrins are acidic proteins, present in all eukaryotes to perform imperative roles in centrosome positioning and segregation. Existing methods for the purification of centrins for biophysical studies involves either multiple steps or yields protein with an affinity tag, which pins additional tag-cleavage step. Therefore, we have made an attempt to develop a simple and single step method for protein purification. We have performed categorical evaluation of existing methods, and describe a one-step procedure based on cleavable Intein-tag, which can be utilized for routine preparation of any isoform of centrins. Since human Centrin-1 and Centrin-2 are devoid of Trp, we exploit this feature to assess the purity of the protein using Tyr fluorescence; an essential point ignored generally. In addition, we report important spectral and hydrodynamic characteristics of human Centrin-1, accounting that HsCentrin-1 has moderate affinity for Ca(2+). Centrin-1 does not gain structure as seen by far- and near-UV circular dichroism, rather there is a loss of ellipticity, though inconsiderable upon binding Ca(2+). PMID:27235176

  19. Addition of equilibrium air to an upwind Navier-Stokes code and other first steps toward a more generalized flow solver

    NASA Technical Reports Server (NTRS)

    Rosen, Bruce S.

    1991-01-01

    An upwind three-dimensional volume Navier-Stokes code is modified to facilitate modeling of complex geometries and flow fields represented by proposed National Aerospace Plane concepts. Code enhancements include an equilibrium air model, a generalized equilibrium gas model and several schemes to simplify treatment of complex geometric configurations. The code is also restructured for inclusion of an arbitrary number of independent and dependent variables. This latter capability is intended for eventual use to incorporate nonequilibrium/chemistry gas models, more sophisticated turbulence and transition models, or other physical phenomena which will require inclusion of additional variables and/or governing equations. Comparisons of computed results with experimental data and results obtained using other methods are presented for code validation purposes. Good correlation is obtained for all of the test cases considered, indicating the success of the current effort.

  20. Assessment of the aerobic preparation and bottom ash addition as pretreatment steps before landfilling: impact on methanogenesis kinetics and leachate parameters.

    PubMed

    Mansour, Alicia A; Motte, Antoine; Pallier, Virginie; Feuillade-Cathalifaud, Geneviève; Ponthieux, Arnaud

    2012-10-01

    This work focuses on assessing the impact of two types of waste pretreatment: addition of bottom ashes and aerobic pretreatment on both the onset and kinetics of methanogenesis and the evolution of different parameters in the leachate. It also studies the correlation between methane production and the different parameters measured in the leachate produced. A total of six 68-L pilots were thus used with fresh municipal solid waste (MSW) shredded to a 40-mm size. After 14 months of landfilling, the control has produced less than 10 NLkg(-1)DM, which corresponds to around 7% of its biochemical methane potential (BMP). Nevertheless, on one hand for aerobically pretreated waste, the lag phase before the onset of methanogenesis is significantly reduced to 0.9 month compared to more than 1 year for the control. In addition to that, on average 110 NLkg(-1)DM (90% of the BMP) is produced within around 6.5 months. On the other hand, the waste with added bottom ash shows a slight improvement of the lag phase over the control for one of the duplicate: 6.1 months of lag phase. At this stage, on average of 26 NLkg(-1)DM waste are detected (22% of the BMP) no final conclusion concerning the impact of bottom ashes could be made. The data obtained for the leachate parameters agrees with the observations on methane production. Statistical correlation study shows that the two components of the corrected PCA interpret 76% of the variability of the data: SUVA (specific UV absorbance at 254 nm) and HPI(*) (% of hydrophilic compounds) are identified as interesting parameters for following up the biodegradation in landfill conditions. PMID:22640801

  1. Three-phase partitioning as a rapid and easy method for the purification and recovery of catalase from sweet potato tubers (Solanum tuberosum).

    PubMed

    Duman, Yonca Avcı; Kaya, Erdem

    2013-07-01

    Three-phase partitioning (TPP) was used to purify and recover catalase from potato crude extract. The method consists of ammonium sulfate saturation, t-butanol addition, and adjustment of pH, respectively. The best catalase recovery (262 %) and 14.1-fold purification were seen in the interfacial phase in the presence of 40 % (w/v) ammonium sulfate saturation with 1.0:1.0 crude extract/t-butanol ratio (v/v) at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the enzyme showed comparatively purification and protein molecular weight was nearly found to be 56 kDa. This study shows that TPP is a simple, economical, and quick method for the recovering of catalase and can be used for the purification process. PMID:23640263

  2. The Borexino purification system

    NASA Astrophysics Data System (ADS)

    Benziger, Jay

    2014-05-01

    Purification of 278 tons of liquid scintillator and 889 tons of buffer shielding for the Borexino solar neutrino detector is performed with a system of combined distillation, water extraction, gas stripping and filtration. The purification system removed K, U and Th by distillation of the pseudocumene solvent and the PPO fluor. Noble gases, Rn, Kr and Ar were removed by gas stripping. Distillation was also employed to remove optical impurities and reduce the attenuation of scintillation light. The success of the purification system has facilitated the first time real time detection of low energy solar neutrinos.

  3. Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3'-ARiBo tag.

    PubMed

    Di Tomasso, Geneviève; Salvail-Lacoste, Alix; Bouvette, Jonathan; Omichinski, James G; Legault, Pascale

    2014-01-01

    Common approaches for purification of RNAs synthesized in vitro by the T7 RNA polymerase often denature the RNA and produce RNAs with chemically heterogeneous 5'- and 3'-ends. Thus, native affinity purification strategies that incorporate 5' and 3' trimming technologies provide a solution to two main disadvantages that arise from standard approaches for RNA purification. This chapter describes procedures for nondenaturing affinity purification of in vitro transcribed RNA using a 3'-ARiBo tag, which yield RNAs with a homogeneous 3'-end. The applicability of the method to RNAs of different sequences, secondary structures, and sizes (29-614 nucleotides) is described, including suggestions for troubleshooting common problems. In addition, this chapter presents three complementary approaches to producing 5'-homogeneity of the affinity-purified RNA: (1) selection of the starting sequence; (2) Cse3 endoribonuclease cleavage of a 5'-CRISPR tag; or (3) self-cleavage of a 5'-hammerhead ribozyme tag. The additional steps to express and purify the Cse3 endonuclease are detailed. In light of recent results, the advantages and limitations of current approaches to achieve 5'-homogeneity of affinity-purified RNA are discussed, such that one can select a suitable strategy to purify the RNA of interest. PMID:25432744

  4. Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

    PubMed Central

    Wysoczynski, Christina L.; Roemer, Sarah C.; Dostal, Vishantie; Barkley, Robert M.; Churchill, Mair E. A.; Malarkey, Christopher S.

    2013-01-01

    Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications. PMID:24013567

  5. Preparative two-dimensional liquid chromatography/mass spectrometry for the purification of complex pharmaceutical samples.

    PubMed

    Zhang, Yinong; Zeng, Lu; Pham, Catherine; Xu, Rongda

    2014-01-10

    A new preparative two-dimensional liquid chromatography/mass spectrometry system (2D LC-LC/MS) has been designed and implemented to enhance capability and resolving power for the separation and purification of pharmaceutical samples. The system was constructed by modifications of a conventional preparative LC/MS instrument with the addition of a set of switching valves and a sample loop, as well as interfacing a custom software program with MassLynx. The system integrates two chromatographic separations from the first and second dimensions into a single automated run to perform the purification of a target compound from a complex mixture without intermediate steps of sample preparation. The chromatography in the first dimension, operated in the heart-cutting mode, separates the target compound from the impurities by mass-triggered fractionation based on its molecular weight. This purified fraction from the first dimension is stored in the sample loop, and then gets transferred to the second column by using at-column dilution. A control software program, coined Prep 2D LCMS, was designed to integrate with MassLynx to retrieve data acquisition status. All of the chromatographic hardware components used in this preparative 2D LC-LC/MS system are from the original open access preparative LC/MS system, which has high level of robustness and affords easy and user-friendly operation. The new system is very versatile and capable of collecting multiple fractions with different masses under various purification modes as configured in the methods, such as conventional one-dimensional (1D) purification and/or 2D purification. This new preparative 2D LC-LC/MS system is therefore the ideal tool for medicinal chemistry lab in drug discovery environment. PMID:24309715

  6. Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing.

    PubMed

    Hou, Ying; Brower, Mark; Pollard, David; Kanani, Dharmesh; Jacquemart, Renaud; Kachuik, Bradley; Stout, James

    2015-01-01

    Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. PMID:26018631

  7. Semiconductor grade, solar silicon purification project

    NASA Technical Reports Server (NTRS)

    Ingle, W. M.; Thompson, S.; Rosler, D.; Jackson, J.

    1977-01-01

    The conversion of metallurgical grade silicon into semiconductor grade silicon by way of a three step SiF2 polymer transport purification process was investigated. Developments in the following areas were also examined: (1) spectroscopic analysis and characterization of (SiF2) sub x polymer and Si sub x F sub y homologue conversion; (2) demonstration runs on the near continuous apparatus; (3) economic analysis; and (4) elemental analysis.

  8. Purification of a Recombinant Polyhistidine-Tagged Glucosyltransferase Using Immobilized Metal-Affinity Chromatography (IMAC).

    PubMed

    de Costa, Fernanda; Barber, Carla J S; Pujara, Pareshkumar T; Reed, Darwin W; Covello, Patrick S

    2016-01-01

    Short peptide tags genetically fused to recombinant proteins have been widely used to facilitate detection or purification without the need to develop specific procedures. In general, an ideal affinity tag would allow the efficient purification of tagged proteins in high yield, without affecting its function. Here, we describe the purification steps to purify a recombinant polyhistidine-tagged glucosyltransferase from Centella asiatica using immobilized metal affinity chromatography. PMID:26843168

  9. Ion exchange purification of scandium

    DOEpatents

    Herchenroeder, Laurie A.; Burkholder, Harvey R.

    1990-10-23

    An improvement in purification of scandium through ion exchange chromatography is disclosed in which the oxidation potential of the eluting solution is altered by the addition of potassium chlorate or ammonium chloride so that removal of contaminants is encouraged. The temperature, pH and concentration of the eluent HEDTA are controlled in order to maintain the scandium in the column while minimizing dilution of the scandium band. Recovery of scandium is improved by pumping dilute scandium over the column prior to stripping the scandium and precipitation. This eliminates the HEDTA ion and other monovalent cations contaminating the scandium band. This method maximizes recovery of scandium while maintaining purity.

  10. Ion exchange purification of scandium

    DOEpatents

    Herchenroeder, L.A.; Burkholder, H.R.

    1990-10-23

    An improvement in purification of scandium through ion exchange chromatography is disclosed in which the oxidation potential of the eluting solution is altered by the addition of potassium chlorate or ammonium chloride so that removal of contaminants is encouraged. The temperature, pH and concentration of the eluent HEDTA are controlled in order to maintain the scandium in the column while minimizing dilution of the scandium band. Recovery of scandium is improved by pumping dilute scandium over the column prior to stripping the scandium and precipitation. This eliminates the HEDTA ion and other monovalent cations contaminating the scandium band. This method maximizes recovery of scandium while maintaining purity. 2 figs.

  11. A DFT study on the NHC catalysed Michael addition of enols to α,β-unsaturated acyl-azoliums. A base catalysed C-C bond-formation step.

    PubMed

    Domingo, Luis R; Sáez, José A; Arnó, Manuel

    2014-02-14

    The NHC catalysed nucleophilic additions of enols to α,β-unsaturated acyl-azolium intermediates have been investigated using DFT methods at the MPWB1K/6-31G** computational level. In the direct and the conjugate additions, formation of a hydrogen bond (HB) with the carboxyl oxygen is not sufficient to favour the C-C bond formation as a consequence of the low nucleophilic character of enols. Interestingly, when enols form a HB with the chloride counterion, the activation energies associated with the conjugate addition decrease as a consequence of the increased nucleophilic character of enols and the increased electrophilic character of the 'acyl-azolium + Cl' ion pair. Analysis of the DFT reactivity indices allows establishing a base catalysed C-C bond-formation step promoted by the chloride counterion. PMID:24343422

  12. High yield soluble bacterial expression and streamlined purification of recombinant human interferon α-2a.

    PubMed

    Bis, Regina L; Stauffer, Tara M; Singh, Surinder M; Lavoie, Thomas B; Mallela, Krishna M G

    2014-07-01

    Interferon α-2a (IFNA2) is a member of the Type I interferon cytokine family, known for its antiviral and anti-proliferative functions. The role of this family in the innate immune response makes it an attractive candidate for the treatment of many viral and chronic immune-compromised diseases. Recombinant IFNA2 is clinically used to modulate hairy cell leukemia as well as hepatitis c. Historically, IFNA2 has been purified from human leukocytes as well as bacterial expression systems. In most cases, bacterial expression of IFNA2 resulted in inclusion body formation, or required numerous purification steps that decreased the protein yield. Here, we describe an expression and purification scheme for IFNA2 using a pET-SUMO bacterial expression system and a single purification step. Using the SUMO protein as the fusion tag achieved high soluble protein expression. The SUMO tag was cleaved with the Ulp1 protease leaving no additional amino acids on the fusion terminus following cleavage. Mass spectrometry, circular dichroism, 2D heteronuclear NMR, and analytical ultracentrifugation confirmed the amino acid sequence identity, secondary and tertiary protein structures, and the solution behavior of the purified IFNA2. The purified protein also had antiviral and anti-proliferative activities comparable to the WHO International Standard, NIBSC 95/650, and the IFNA2 standard available from PBL Assay Science. Combining the expression and purification protocols developed here to produce IFNA2 on a laboratory scale with the commercial fermenter technology commonly used in pharmaceutical industry may further enhance IFNA2 yields, which will promote the development of interferon-based protein drugs to treat various disorders. PMID:24794500

  13. The development, characterization, and demonstration of a novel strategy for purification of recombinant proteins expressed in plants.

    PubMed

    Tremblay, Reynald; Diao, Hong; Huner, Norm; Jevnikar, Anthony M; Ma, Shengwu

    2011-12-01

    Plants have attracted increasing attention as an expression platform for the production of pharmaceutical proteins due to its unlimited scalability and low cost potential. However, compared to other expression systems, plants accumulate relatively low levels of foreign proteins, thus necessitating the development of efficient systems for purification of foreign proteins from plant tissues. We have developed a novel strategy for purification of recombinant proteins expressed in plants, based on genetic fusion to soybean agglutinin (SBA), a homotetrameric lectin that binds to N-acetyl-D-galactosamine. Previously it was shown that high purity SBA could be recovered from soybean with an efficiency of greater than 90% following one-step purification using N-acetyl-D-galactosamine-agar columns. We constructed an SBA fusion protein containing the reporter green fluorescent protein (GFP) and transiently expressed it in N. benthamiana plants. We achieved over 2.5% of TSP accumulation in leaves of N. benthamiana. Confocal microscopic analysis demonstrated in vivo activity of the fused GFP partner. Importantly, high purity rSBA-GFP was recovered from crude leaf extract with ~90% yield via one-step purification on N-acetyl-D-galactosamine-agar columns, and the purified fusion protein was able to induce the agglutination of rabbit red blood cells. Combined with this, tetrameric assembly of the fusion protein was demonstrated via western blotting. In addition, rSBA-GFP retained its GFP signal on agglutinated red blood cells, demonstrating the feasibility of using rSBA-GFP for discrimination of cells that bear the ligand glycan on their surface. This work validates SBA as an effective affinity tag for simple and rapid purification of genetically fused proteins. PMID:21365323

  14. Surface processes during purification of InP quantum dots

    PubMed Central

    Emelin, Pavel; Vinokurov, Alexander; Dorofeev, Sergey; Abakumov, Artem; Kuznetsova, Tatiana

    2014-01-01

    Summary Recently, a new simple and fast method for the synthesis of InP quantum dots by using phosphine as phosphorous precursor and myristic acid as surface stabilizer was reported. Purification after synthesis is necessary to obtain samples with good optical properties. Two methods of purification were compared and the surface processes which occur during purification were studied. Traditional precipitation with acetone is accompanied by a small increase in photoluminescence. It occurs that during the purification the hydrolysis of the indium precursor takes place, which leads to a better surface passivation. The electrophoretic purification technique does not increase luminescence efficiency but yields very pure quantum dots in only a few minutes. Additionally, the formation of In(OH)3 during the low temperature synthesis was explained. Purification of quantum dots is a very significant part of postsynthetical treatment that determines the properties of the material. But this subject is not sufficiently discussed in the literature. The paper is devoted to the processes that occur at the surface of quantum dots during purification. A new method of purification, electrophoresis, is investigated and described in particular. PMID:25161857

  15. Production of 7α,15α-diOH-DHEA from dehydroepiandrosterone by Colletotrichum lini ST-1 through integrating glucose-feeding with multi-step substrate addition strategy.

    PubMed

    Li, Cong; Li, Hui; Sun, Jin; Zhang, XinYue; Shi, Jinsong; Xu, Zhenghong

    2016-08-01

    Hydroxylation of dehydroepiandrosterone (DHEA) to 3β,7α,15α-trihydroxy-5-androstene-17-one (7α,15α-diOH-DHEA) by Colletotrichum lini ST-1 is an essential step in the synthesis of many steroidal drugs, while low DHEA concentration and 7α,15α-diOH-DHEA production are tough problems to be solved urgently in industry. In this study, the significant improvement of 7α,15α-diOH-DHEA yield in 5-L stirred fermenter with 15 g/L DHEA was achieved. To maintain a sufficient quantity of glucose for the bioconversion, glucose of 15 g/L was fed at 18 h, the 7α,15α-diOH-DHEA yield and dry cell weight were increased by 17.7 and 30.9 %, respectively. Moreover, multi-step DHEA addition strategy was established to diminish DHEA toxicity to C. lini, and the 7α,15α-diOH-DHEA yield raised to 53.0 %. Further, a novel strategy integrating glucose-feeding with multi-step addition of DHEA was carried out and the product yield increased to 66.6 %, which was the highest reported 7α,15α-diOH-DHEA production in 5-L stirred fermenter. Meanwhile, the conversion course was shortened to 44 h. This strategy would provide a possible way in enhancing the 7α,15α-diOH-DHEA yield in pharmaceutical industry. PMID:27094679

  16. Purification of aqueous cellulose ethers

    SciTech Connect

    Bartscherer, K.A.; de Pablo, J.J.; Bonnin, M.C.; Prausnitz, J.M.

    1990-07-01

    Manufacture of cellulose ethers usually involves high amounts of salt by-products. For application of the product, salt must be removed. In this work, we have studied the injection of high-pressure CO{sub 2} into an aqueous polymer-salt solution; we find that upon addition of isopropanol in addition to CO{sub 2}, the solution separates into two phases. One phase is rich in polymer and water, and the other phase contains mostly isopropanol, water and CO{sub 2}. The salt distributes between the two phases, thereby offering interesting possibilities for development of a new purification process for water-soluble polymers. This work presents experimental phase-equilibrium data for hydroxyethyl cellulose and sodium carboxymethyl cellulose with sodium acetate and potassium sulfate, respectively, in the region 40{degree}C and 30 to 80 bar. Based on these data, we suggest a process for the manufacture and purification of water-soluble cellulose ethers. 15 refs., 14 figs., 9 tabs.

  17. Purification of genuine multipartite entanglement

    SciTech Connect

    Huber, Marcus; Plesch, Martin

    2011-06-15

    In tasks where multipartite entanglement plays a central role, state purification is, due to inevitable noise, a crucial part of the procedure. We consider a scenario exploiting the multipartite entanglement in a straightforward multipartite purification algorithm and compare it to bipartite purification procedures combined with state teleportation. While complete purification requires an infinite amount of input states in both cases, we show that for an imperfect output fidelity the multipartite procedure exhibits a major advantage in terms of input states used.

  18. [Extraction and purification method of rice DNA from rice powder containing Konjak flour].

    PubMed

    Minematsu, Kazuhiko; Nakamura, Kosuke; Akiyama, Hiroshi; Harikai, Naoki; Nakajima, Osamu; Kitta, Kazumi; Teshima, Reiko; Iizuka, Tayoshi

    2010-01-01

    Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR. PMID:21071909

  19. Affinity purification of aprotinin from bovine lung.

    PubMed

    Xin, Yu; Liu, Lanhua; Chen, Beizhan; Zhang, Ling; Tong, Yanjun

    2015-05-01

    An affinity protocol for the purification of aprotinin from bovine lung was developed. To simulate the structure of sucrose octasulfate, a natural specific probe for aprotinin, the affinity ligand was composed of an acidic head and a hydrophobic stick, and was then linked with Sepharose. The sorbent was then subjected to adsorption analysis with pure aprotinin. The purification process consisted of one step of affinity chromatography and another step of ultrafiltration. Then purified aprotinin was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, trypsin inhibitor activity, gel-filtration, and thin-layer chromatography analysis. As calculated, the theoretical maximum adsorption (Qmax ) of the affinity sorbent was 25,476.0 ± 184.8 kallikrein inactivator unit/g wet gel; the dissociation constant of the complex "immobilized ligand-aprotinin" (Kd ) was 4.6 ± 0.1 kallikrein inactivator unit/mL. After the affinity separation of bovine lung aprotinin, reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and gel-filtration chromatography revealed that the protein was a single polypeptide, and the purities were ∼ 97 and 100%, respectively; the purified peptide was also confirmed with aprotinin standard by gel-filtration chromatography and thin-layer chromatography. After the whole purification process, protein, and bioactivity recoveries were 2.2 and 92.6%, respectively; and the specific activity was up to 15,907.1 ± 10.2 kallikrein inactivator unit/mg. PMID:25677462

  20. Growth of Thin, Anisotropic, π-Conjugated Molecular Films by Stepwise "Click" Assembly of Molecular Building Blocks: Characterizing Reaction Yield, Surface Coverage, and Film Thickness versus Addition Step Number.

    PubMed

    Demissie, Abel T; Haugstad, Greg; Frisbie, C Daniel

    2015-07-15

    We report the systematic characterization of anisotropic, π-conjugated oligophenyleneimine (OPI) films synthesized using stepwise imine condensation, or "click" chemistry. Film synthesis began with a self-assembled monolayer (SAM) of 4-formylthiophenol or 4-aminothiophenol on Au, followed by repetitive, alternate addition of terephthalaldehyde (benzene-1,4-dicarbaldehyde) or 1,4-benzenediamine to form π-conjugated films ranging from 0.6-5.5 nm in thickness. By systematically capping the OPI films with a redox or halogen label, we were able to measure the relative surface coverage after each monomer addition via Rutherford backscattering spectrometry, X-ray photoelectron spectroscopy, spectroscopic ellipsometry, reflection-absorption infrared spectroscopy, and cyclic voltammetry. Nuclear reaction analysis was also employed for the first time on a SAM to calculate the surface coverage of carbon atoms after each stepwise addition. These six different analysis methods indicate that the average extent of reaction is 99% for each addition step. The high yield and molecular surface coverage confirm the efficacy of Schiff base chemistry, at least with the terephthalaldehyde and 1,4-benzenediamine monomers, for preparing high-quality molecular films with π conjugation normal to the substrate. PMID:26098519

  1. Water purification in Borexino

    SciTech Connect

    Giammarchi, M.; Balata, M.; Ioannucci, L.; Nisi, S.; Goretti, A.; Ianni, A.; Miramonti, L.

    2013-08-08

    Astroparticle Physics and Underground experiments searching for rare nuclear events, need high purity materials to act as detectors or detector shielding. Water has the advantage of being cheap, dense and easily available. Most of all, water can be purified to the goal of obatining a high level of radiopurity. Water Purification can be achieved by means of a combination of processes, including filtration, reverse osmosis, deionization and gas stripping. The Water Purification System for the Borexino experiment, will be described together with its main performances.

  2. High efficiency production and purification of 86Y based on electrochemical separation.

    PubMed

    Lukić, Dragoljub; Tamburella, Claire; Buchegger, Franz; Beyer, Gerd-Jürgen; Comor, Jozef J; Seimbille, Yann

    2009-04-01

    As an intermediate half-life positron emitter (86)Y is an attractive radioisotope for positron emission tomography (PET) studies, particularly for patient specific dosimetry planning of (90)Y-based radiotherapy procedures. It can be conveniently produced by a small-sized cyclotron via the (86)Sr(p,n)(86)Y nuclear reaction. The optimization of the electrochemical separation of (86)Y from the target material and its purification was done by modeling the whole production cycle using (90)Y. The radionuclide was isolated using four electrodes in two electrolytic steps. In the first step two Pt plate anodes and a Pt Winkler cathode were used and the electro-deposition yield was determined in constant current mode of operation. In addition, the influence of pH on the efficiency of this first step was investigated. The second electrolysis, with Winkler electrode as anode and a Pt wire as cathode, was also performed in constant current mode of operation. The kinetics of recovery of the deposited activity on the Pt wire was investigated in acidic solutions. The optimized electrochemical method was then applied for (86)Y separation and purification. This modified procedure was proved to be faster and simpler than the previously proposed electrochemical techniques and is more convenient for automation of the routine production of (86)Y. PMID:19181533

  3. Purification of goat beta-lactoglobulin from whey by an ultrafiltration membrane enzymic reactor.

    PubMed

    Sannier, F; Bordenave, S; Piot, J M

    2000-02-01

    This paper presents a novel contribution to the purification of goat beta-lactoglobulin by using an ultrafiltration membrane enzymic reactor. The basis of the purification process was the enzymic hydrolysis of contaminating proteins, alpha-lactalbumin and traces of serum albumin, by pepsin at 40 degrees C and pH 2, conditions under which beta-lactoglobulin is resistant to peptic digestion. Simultaneously, beta-lactoglobulin and peptides were separated by ultrafiltration. beta-Lactoglobulin was retained in the reactor while peptides generated by hydrolysis from alpha-lactalbumin and serum albumin permeated through the membrane. The process was made continuous by the addition of fresh whey to replace the lost permeate. Three mineral membranes with 10, 30 and 50 kDa molecular mass cut-off were tested and the 30 kDa membrane was selected for the continuous process. The simultaneous purification and concentration of beta-lactoglobulin from clarified goats' whey was achieved in a single step. The ultrafiltration membrane enzymic reactor could treat eight reactor volumes of clarified whey. The recovery of beta-lactoglobulin was 74%, its purity was 84% and its concentration 6.6-fold that in the initial clarified whey. PMID:10717842

  4. Purification of recombinant proteins from mammalian cell culture using a generic double-affinity chromatography scheme.

    PubMed

    Cass, Brian; Pham, Phuong Lan; Kamen, Amine; Durocher, Yves

    2005-03-01

    Transient transfection of mammalian cells has proven to be a useful technique for the rapid production of recombinant proteins because of its ability to produce milligram quantities within 2 weeks following cloning of their corresponding cDNA. This rapid production also requires a fast and efficient purification scheme that can be applied generically, typically through the use of affinity tags such as the polyhistidine-tag for capture by immobilized metal-affinity chromatography (IMAC) or the Strep-tag II, which binds to the StrepTactin affinity ligand. However, one-step purification using either of these tags has disadvantages in terms of yield, elution conditions, and purity. Here, we show that the addition of both Strep-tag-II and (His)(8) to the C-terminal of r-proteins allows efficient purification by consecutive IMAC and StrepTactin affinity. This approach has been successfully demonstrated using the intracellular protein DsRed, as well as two secreted proteins, secreted alkaline phosphatase (SEAP) and vascular endothelial growth factor (VEGF), all produced by transient transfection of HEK293-EBNA1 cells in medium supplemented with bovine calf serum. All proteins were purified to >99% homogeneity with yields varying from 29 to 81%. PMID:15721774

  5. Isolation and Purification of Cu-free Methanobactin from Methylosinus trichosporium OB3b

    SciTech Connect

    M Pesch; I Christl; K Barmettler; S Kraemer; R Kretzschmar

    2011-12-31

    The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin.

  6. A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana.

    PubMed

    Stotz, Henrik U; Findling, Simone; Nukarinen, Ella; Weckwerth, Wolfram; Mueller, Martin J; Berger, Susanne

    2014-01-01

    Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes. PMID:25482810

  7. Detergent-Free Membrane Protein Purification.

    PubMed

    Rothnie, Alice J

    2016-01-01

    Membrane proteins are localized within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilizing or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilizing protein structure, is time consuming and expensive. Here we present a recently-developed method using a styrene-maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment. PMID:27485341

  8. Monogamy, polygamy, and other properties of entanglement of purification

    NASA Astrophysics Data System (ADS)

    Bagchi, Shrobona; Pati, Arun Kumar

    2015-04-01

    For bipartite pure and mixed quantum states, in addition to the quantum mutual information, there is another measure of total correlation, namely, the entanglement of purification. We study the monogamy, polygamy, and additivity properties of the entanglement of purification for pure and mixed states. In this paper, we show that, in contrast to the quantum mutual information which is strictly monogamous for any tripartite pure states, the entanglement of purification is polygamous for the same. This shows that there can be genuinely two types of total correlation across any bipartite cross in a pure tripartite state. Furthermore, we find the lower bound and actual values of the entanglement of purification for different classes of tripartite and higher-dimensional bipartite mixed states. Thereafter, we show that if entanglement of purification is not additive on tensor product states, it is actually subadditive. Using these results, we identify some states which are additive on tensor products for entanglement of purification. The implications of these findings on the quantum advantage of dense coding are briefly discussed, whereby we show that for tripartite pure states, it is strictly monogamous and if it is nonadditive, then it is superadditive on tensor product states.

  9. Design of underwater superoleophobic TiO2 coatings with additional photo-induced self-cleaning properties by one-step route bio-inspired from fish scales

    NASA Astrophysics Data System (ADS)

    Wang, Hao; Guo, Zhiguang

    2014-05-01

    Self-cleaning properties inspired by the structures and functions of some creatures are of great interest since the late 20th century. In this paper, TiO2 coatings with hierarchical rutile TiO2 flowers on fluorine-doped tin oxide substrate are fabricated through a simple one-step hydrothermal method. The flower-like coatings exhibit superhydrophilicity in air and superoleophobicity underwater with a contact angle as high as 157°, presenting good underwater self-cleaning performance. In addition, when contaminated by oleic acid, the as-prepared TiO2 coatings also exhibit excellent photocatalytic capability under ultraviolet irradiation, which demonstrated self-cleaning properties in a different way. This self-cleaning film provides a good strategy for some industrial and ocean applications.

  10. Design of underwater superoleophobic TiO{sub 2} coatings with additional photo-induced self-cleaning properties by one-step route bio-inspired from fish scales

    SciTech Connect

    Wang, Hao; Guo, Zhiguang

    2014-05-05

    Self-cleaning properties inspired by the structures and functions of some creatures are of great interest since the late 20th century. In this paper, TiO{sub 2} coatings with hierarchical rutile TiO{sub 2} flowers on fluorine-doped tin oxide substrate are fabricated through a simple one-step hydrothermal method. The flower-like coatings exhibit superhydrophilicity in air and superoleophobicity underwater with a contact angle as high as 157°, presenting good underwater self-cleaning performance. In addition, when contaminated by oleic acid, the as-prepared TiO{sub 2} coatings also exhibit excellent photocatalytic capability under ultraviolet irradiation, which demonstrated self-cleaning properties in a different way. This self-cleaning film provides a good strategy for some industrial and ocean applications.

  11. A Quick and Efficient Method for the Purification of Endoderm Cells Generated from Human Embryonic Stem Cells.

    PubMed

    Davenport, Claudia; Diekmann, Ulf; Naujok, Ortwin

    2016-01-01

    The differentiation capabilities of pluripotent stem cells such as embryonic stem cells (ESCs) allow a potential therapeutic application for cell replacement therapies. Terminally differentiated cell types could be used for the treatment of various degenerative diseases. In vitro differentiation of these cells towards tissues of the lung, liver and pancreas requires as a first step the generation of definitive endodermal cells. This step is rate-limiting for further differentiation towards terminally matured cell types such as insulin-producing beta cells, hepatocytes or other endoderm-derived cell types. Cells that are committed towards the endoderm lineage highly express a multitude of transcription factors such as FOXA2, SOX17, HNF1B, members of the GATA family, and the surface receptor CXCR4. However, differentiation protocols are rarely 100% efficient. Here, we describe a method for the purification of a CXCR4+ cell population after differentiation into the DE by using magnetic microbeads. This purification additionally removes cells of unwanted lineages. The gentle purification method is quick and reliable and might be used to improve downstream applications and differentiations. PMID:26966833

  12. Development of robust antibody purification by optimizing protein-A chromatography in combination with precipitation methodologies.

    PubMed

    Chollangi, Srinivas; Parker, Ray; Singh, Nripen; Li, Yi; Borys, Michael; Li, Zhengjian

    2015-11-01

    To be administered to patients, therapeutic monoclonal antibodies must have very high purity, with process related impurities like host-cell proteins (HCPs) and DNA reduced to <100 ppm and <10 ppb, respectively, relative to desired product. Traditionally, Protein-A chromatography as a capture step has been the work horse for clearing a large proportion of these impurities. However, remaining levels of process and product related impurities still present significant challenges on the development of polishing steps further downstream. In this study, we have incorporated high throughput screening to evaluate three areas of separation: (i) Harvest treatment; (ii) Protein-A Chromatography; and (iii) Low pH Viral Inactivation. Precipitation with low pH treatment of cell culture harvest resulted in selective removal of impurities while manipulating the pH of wash buffers used in Protein-A chromatography and incorporating wash additives that disrupt various modes of protein-protein interaction resulted in further and more pronounced reduction in impurity levels. In addition, our study also demonstrate that optimizing the neutralization pH post Protein-A elution can result in selective removal of impurities. When applied over multiple mAbs, this optimization method proved to be very robust and the strategy provides a new and improved purification process that reduces process related impurities like HCPs and DNA to drug substance specifications with just one chromatography column and open avenues for significant decrease in operating costs in monoclonal antibody purification. PMID:25950654

  13. PHOEBE - step by step manual

    NASA Astrophysics Data System (ADS)

    Zasche, P.

    2016-03-01

    An easy step-by-step manual of PHOEBE is presented. It should serve as a starting point for the first time users of PHOEBE analyzing the eclipsing binary light curve. It is demonstrated on one particular detached system also with the downloadable data and the whole procedure is described easily till the final trustworthy fit is being reached.

  14. Step Pultrusion

    NASA Astrophysics Data System (ADS)

    Langella, A.; Carbone, R.; Durante, M.

    2012-12-01

    The pultrusion process is an efficient technology for the production of composite material profiles. Thanks to this positive feature, several studies have been carried out, either to expand the range of products made using the pultrusion technology, or improve its already high production rate. This study presents a process derived from the traditional pultrusion technology named "Step Pultrusion Process Technology" (SPPT). Using the step pultrusion process, the final section of the composite profiles is obtainable by means of a progressive cross section increasing through several resin cure stations. This progressive increasing of the composite cross section means that a higher degree of cure level can be attained at the die exit point of the last die. Mechanical test results of the manufactured pultruded samples have been used to compare both the traditional and the step pultrusion processes. Finally, there is a discussion on ways to improve the new step pultrusion process even further.

  15. [Purification of enramycin by macroporous resin adsorption and reversed phase chromatography purification].

    PubMed

    Jiaxin, Wu; Yongdong, Huang; Peng, Qi; Jihong, He; Ping, Li; Guodong, Zhang; Meixian, Zhao

    2014-11-01

    Enramycin is a polypeptide antibiotic and new, safe animal feed additive. A new purification process was developed, based on pre-purification by macroporous resin and refining by reversed phase chromatography. AB-8 macroporous resin was used for the pre-purification process of enramycin, with an elution buffer of 0.012 mol/L aqueous HCl solution-methanol (50: 50, V/V). Then, enramycin a and enramycin b were separated effectively by C18 reversed phase chromatography, with a elution buffer of 0.05 mol/L aqueous KH2PO4 solution-acetonitrile (70: 30, V/V, pH 4.5). The purities of enramycin a and enramycin b were up to 98.5% and 98.0%, respectively. The yield reached 29.2%. This study would provide a useful reference for the preparation of enramycin a and enramycin b with a high purity. PMID:25985521

  16. Purification and concentration of DNA from aqueous solutions.

    PubMed

    Moore, D

    2001-05-01

    This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. Phenol extraction with ethanol precipitation is described along with an alternate procedure of precipitating DNA with isopropanol. A Support Protocol describes concentration of DNA using butanol. PMID:18429080

  17. Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.

    PubMed

    Demir, Hülya; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

    2003-02-01

    In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots. PMID:12693814

  18. Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography.

    PubMed

    Pato, Tânia Pinheiro; Barbosa, Antonio de Pádua R; da Silva Junior, José Godinho

    2006-03-01

    Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry. PMID:16469547

  19. Purification of GST-Tagged Proteins.

    PubMed

    Schäfer, Frank; Seip, Nicole; Maertens, Barbara; Block, Helena; Kubicek, Jan

    2015-01-01

    This protocol describes the purification of recombinant proteins fused to glutathione S-transferase (GST, GST-tagged proteins) by Glutathione Affinity purification. The GST tag frequently increases the solubility of the fused protein of interest and thus enables its purification and subsequent functional characterization. The GST-tagged protein specifically binds to glutathione immobilized to a matrix (e.g., agarose) and can be easily separated from a cell lysate by a bind-wash-elute procedure. GST-tagged proteins are often used to study protein-protein interactions, again making use of glutathione affinity in a procedure called a GST pull-down assay. The protocol is designed to process 200 ml of E. coli culture expressing intermediate to high amounts of a GST-tagged protein (~25 mg l(-1)). Depending on the expression rate or the available culture volume, the scale can be increased or decreased linearly. The protocol can also be used to purify GST-tagged proteins from other expression systems, such as insect or mammalian cells. Tips are provided to aid in modifying certain steps if proteins shall be recovered from alternative expression systems. PMID:26096507

  20. Ethanol precipitation for purification of recombinant antibodies.

    PubMed

    Tscheliessnig, Anne; Satzer, Peter; Hammerschmidt, Nikolaus; Schulz, Henk; Helk, Bernhard; Jungbauer, Alois

    2014-10-20

    Currently, the golden standard for the purification of recombinant humanized antibodies (rhAbs) from CHO cell culture is protein A chromatography. However, due to increasing rhAbs titers alternative methods have come into focus. A new strategy for purification of recombinant human antibodies from CHO cell culture supernatant based on cold ethanol precipitation (CEP) and CaCl2 precipitation has been developed. This method is based on the cold ethanol precipitation, the process used for purification of antibodies and other components from blood plasma. We proof the applicability of the developed process for four different antibodies resulting in similar yield and purity as a protein A chromatography based process. This process can be further improved using an anion-exchange chromatography in flowthrough mode e.g. a monolith as last step so that residual host cell protein is reduced to a minimum. Beside the ethanol based process, our data also suggest that ethanol could be replaced with methanol or isopropanol. The process is suited for continuous operation. PMID:25087738

  1. Affinity Purification of Antibodies.

    PubMed

    Hnasko, Robert M; McGarvey, Jeffery A

    2015-01-01

    Antibodies are provided in a variety of formats that include antiserum, hybridoma culture supernatant, or ascites. They can all be used successfully in crude form for the detection of target antigens by immunoassay. However, it is advantageous to use purified antibody in defined quantity to facilitate assay reproducibility, economy, and reduced interference of nonspecific components as well as improved storage, stability, and bio-conjugation. Although not always necessary, the relative simplicity of antibody purification using commercially available protein-A, protein-G, or protein-L resins with basic chromatographic principles warrants purification when antibody source material is available in sufficient quantity. Here, we define three simple methods using immobilized (1) protein-A, (2) protein-G, and (3) protein-L agarose beads to yield highly purified antibody. PMID:26160561

  2. High-throughput Protein Purification and Quality Assessment for Crystallization

    PubMed Central

    Kim, Youngchang; Babnigg, Gyorgy; Jedrzejczak, Robert; Eschenfeldt, William H.; Li, Hui; Maltseva, Natalia; Hatzos-Skintges, Catherine; Gu, Minyi; Makowska-Grzyska, Magdalena; Wu, Ruiying; An, Hao; Chhor, Gekleng; Joachimiak, Andrzej

    2012-01-01

    The ultimate goal of structural biology is to understand the structural basis of proteins in cellular processes. In structural biology, the most critical issue is the availability of high-quality samples. “Structural biology-grade” proteins must be generated in the quantity and quality suitable for structure determination using X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. The purification procedures must reproducibly yield homogeneous proteins or their derivatives containing marker atom(s) in milligram quantities. The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. With structural genomics emphasizing a genome-based approach in understanding protein structure and function, a number of unique structures covering most of the protein folding space have been determined and new technologies with high efficiency have been developed. At the Midwest Center for Structural Genomics (MCSG), we have developed semi-automated protocols for high-throughput parallel protein expression and purification. A protein, expressed as a fusion with a cleavable affinity tag, is purified in two consecutive immobilized metal affinity chromatography (IMAC) steps: (i) the first step is an IMAC coupled with buffer-exchange, or size exclusion chromatography (IMAC-I), followed by the cleavage of the affinity tag using the highly specific Tobacco Etch Virus (TEV) protease; [1] the second step is IMAC and buffer exchange (IMAC-II) to remove the cleaved tag and tagged TEV protease. These protocols have been implemented on multidimensional chromatography workstations and, as we have shown, many proteins can be successfully produced in large-scale. All methods and protocols used for purification, some developed by MCSG, others adopted and integrated into the MCSG purification pipeline and more recently the Center for Structural Genomics of Infectious Diseases (CSGID) purification pipeline, are

  3. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1992-01-01

    A water purification/recycling system developed by Photo-Catalytics, Inc. (PCI) for NASA is commercially available. The system cleanses and recycles water, using a "photo-catalysis" process in which light or radiant energy sparks a chemical reaction. Chemically stable semiconductor powders are added to organically polluted water. The powder absorbs ultraviolet light, and pollutants are oxidized and converted to carbon dioxide. Potential markets for the system include research and pharmaceutical manufacturing applications, as well as microchip manufacture and wastewater cleansing.

  4. Effect of chlorine purification on oxidation resistance of some mechanical carbons

    NASA Technical Reports Server (NTRS)

    Wisander, D. W.; Allen, G. P.

    1974-01-01

    Oxidation experiments were conducted with some experimental and commercial mechanical carbons at 650 C in dry air flowing at 28 cc/sec (STP). In general, purification of these carbon-graphites with chlorine at 2800 C improved oxidation resistance. Additional improvements in oxidation resistance were obtained from purification followed by an antioxidant (zinc phosphate) treatment. For the commercial materials, purification alone gave greater oxidation resistance than the antioxidant treatment alone. The reverse, however, was the case for the experimental materials.

  5. Californium purification and electrodeposition

    SciTech Connect

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of the feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.

  6. Californium purification and electrodeposition

    DOE PAGESBeta

    Burns, Jonathan D.; Van Cleve, Shelley M.; Smith, Edward Hamilton; Boll, Rose Ann

    2014-11-30

    The staff at the Radiochemical Engineering Development Center, located at Oak Ridge National Laboratory, produced a 6.3 ± 0.4 GBq (1.7 ± 0.1 Ci) 252Cf source for the Californium Rare Isotope Breeder Upgrade (CARIBU) project at Argonne National Laboratory’s Argonne Tandem Linac Accelerator System. The source was produced by electrodeposition of a 252Cf sample onto a stainless steel substrate, which required material free from excess mass for efficient deposition. The resulting deposition was the largest reported 252Cf electrodeposition source ever produced. Several different chromatographic purification methods were investigated to determine which would be most effective for final purification of themore » feed material used for the CARIBU source. The separation of lanthanides from the Cf was of special concern. Furthermore, the separation, using 145Sm, 153Gd, and 249Cf as tracers, was investigated using BioRad AG 50X8 in α-hydroxyisobutyric acid, Eichrom LN resin in both HNO3 and HCl, and Eichrom TEVA resin in NH4SCN. The TEVA NH4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

  7. Probabilistic theories with purification

    SciTech Connect

    Chiribella, Giulio; D'Ariano, Giacomo Mauro; Perinotti, Paolo

    2010-06-15

    We investigate general probabilistic theories in which every mixed state has a purification, unique up to reversible channels on the purifying system. We show that the purification principle is equivalent to the existence of a reversible realization of every physical process, that is, to the fact that every physical process can be regarded as arising from a reversible interaction of the system with an environment, which is eventually discarded. From the purification principle we also construct an isomorphism between transformations and bipartite states that possesses all structural properties of the Choi-Jamiolkowski isomorphism in quantum theory. Such an isomorphism allows one to prove most of the basic features of quantum theory, like, e.g., existence of pure bipartite states giving perfect correlations in independent experiments, no information without disturbance, no joint discrimination of all pure states, no cloning, teleportation, no programming, no bit commitment, complementarity between correctable channels and deletion channels, characterization of entanglement-breaking channels as measure-and-prepare channels, and others, without resorting to the mathematical framework of Hilbert spaces.

  8. Automated two-step chromatography using an ÄKTA equipped with in-line dilution capability.

    PubMed

    Winters, Dwight; Chu, Carolyn; Walker, Kenneth

    2015-12-11

    There has been a great emphasis on developing higher-throughput protein purification techniques to screen potential human therapeutics faster and more efficiently. Not only is it desirable to have high-throughput purification for initial screens but it is also desirable to efficiently purify selected protein therapeutics in the amounts and purity required for definitive assays. Current automated tandem technologies involve size exclusion as a second step that often fails to generate the required purity, is not robust and can only be operated at a limited scale. We have modified an ÄKTA to enable in-line dilution, assuring that the automated loading of a second column from a first column elution can be modified to a pH and ionic strength which is suitable for binding to the second column. For example, Protein A can be employed as a first step followed by direct loading on to a cation exchange column by conditioning the Protein A elution using the in-line diluter. Using this method as described, up to six samples of 1L each can be purified through two columns without human intervention per day per machine, and the system produces good yields of purified protein over a wide range of loading levels (12-300mg). In addition, the system employs guanidine HCl regeneration, followed by a sodium hydroxide wash between purification runs, minimizing the possibility of carryover contamination. The system is described at the 5mL and the 10mL column sizes; however, it could readily be programed for 100mL columns to enable larger-scale purifications. Using this system to automate two-column purifications minimizes human intervention, increases efficiency and minimizes the risk of human error. PMID:26596873

  9. Ammonia as a preferred additive in chiral and achiral applications of supercritical fluid chromatography for small, drug-like molecules.

    PubMed

    Ventura, Manuel; Murphy, Brent; Goetzinger, Wolfgang

    2012-01-13

    Supercritical fluid chromatography is routinely utilized by analytical separations groups in the pharmaceutical industry to efficiently handle separations for discovery medicinal chemistry purposes. Purifications are performed on samples ranging from a few milligrams up to hundreds of grams. Basic additives dissolved into the liquid component of the SFC mobile phase are commonly used to improve peak shape and efficiency in achiral and chiral separations. While for purposes of analysis there is minimal consequence to additive introduction in the mobile phase, for preparative separations one needs to consider the potential effect of an additive's presence when concentrated with the desired compound. Following an SFC purification using an additive-containing modifier, the resulting fractions will contain an easily evaporated modifier, and after its evaporation perhaps still significant levels of the less volatile additive. Depending on the aqueous solubility and basicity of the final product, the process of removing basic amine additives can be time-consuming and can result in reduced yields. NMR analysis following preparative isolation and evaporation often reveals the fact of insufficient removal of the chromatographic additive even after aqueous work up steps. In this study, ammonia is evaluated as an alternative additive to strong bases such as diethylamine (DEA) in SFC purification and analysis and to the authors' knowledge no previous publication has been written describing the application of methanolic ammonia as an additive for SFC separations. Dimethylethylamine (DMEA), a more volatile additive than DEA, is also evaluated relative to ammonia for its potential to simplify the isolation process after purification and in terms of chromatographic performance. The loss in concentration of ammonia in methanol modifier over time due to evaporation and effects of that loss are also described. Furthermore, for ammonia the analytical benefit is shown to extend to on

  10. Foaming of proteins: New prospects for enzyme purification processes.

    PubMed

    Linke, D; Berger, R G

    2011-04-10

    Efficient techniques for the isolation of enzymes from a microbial production culture are required to meet the growing needs of the "White Biotechnologies" for novel catalysts. Traditional protein purification procedures typically comprise multistep operations, which inevitably come along with significant losses of enzyme activity. Foaming offers an alternative minimizing the processing steps, preserving the purification efficiency and decreasing the activity losses all at the same time. This review provides an insight into the foaming process itself and its application in separating enzymes from model systems and from complex media, such as microbial cultures. Examples demonstrate fractionated foaming and the tweezer technique. PMID:20670663

  11. Cloning, soluble expression, and purification of the RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae.

    PubMed

    Chhetri, Gaurav; Ghosh, Arabinda; Chinta, Ramesh; Akhtar, Sohail; Tripathi, Timir

    2015-01-01

    We report the molecular cloning, expression, and single-step homogeneous purification of RNA polymerase II subunit RPB5 from Saccharomyces cerevisiae. RPB5 is a 210 amino acid nuclear protein that functions as the fifth largest subunit of polymerase II and plays a central role in transcription. The gene that codes for RPB5 was generated by amplification by polymerase chain reaction. It was then inserted in the expression vector pET28a(+) under the transcriptional control of the bacteriophage T7 promoter and lac operator. BL21(DE3) Escherichia coli strain transformed with the rpb5 expression vector pET28a(+)-rpb5 accumulates large amounts of a soluble protein of about 30 kDa (25 kDa plus 5 kDa double His6-Tag at N and C-terminal). The protein was purified to homogeneity using immobilized metal affinity chromatography. RPB5 recombinant protein was further confirmed by immunoblotting with anti-His antibody. In this study, the expression and purification procedures have provided a simple and efficient method to obtain pure RPB5 in large quantities. This will provide an opportunity to study the role of S. cerevisiae RPB5 in gene expression and transcription regulation. Furthermore, it can provide additional knowledge of the interaction partners of RPB5 during various steps of transcription and gene expression. PMID:25551420

  12. Electron nanoprobe induced oxidation: a simulation of direct-write purification.

    PubMed

    Fowlkes, J D; Geier, B; Lewis, B B; Rack, P D; Stanford, M G; Winkler, R; Plank, H

    2015-07-28

    Electron beam direct-write has recently taken a large step forward with the advent of methods to purify deposits. This development has opened the door for future direct-write device prototyping and editing. In one such approach, an additional beam scanning procedure removes carbonaceous impurities via oxidation from metal-carbon deposits (e.g., PtC5) in the presence of H2O or O2 after deposition. So far, critical aspects of the oxidation reaction remain unclear; experiments reveal clearly that electron stimulated oxidation drives the process yet it is not understood why H2O purifies by a bottom-up mechanism while O2 purifies from the top-down. The simulation results presented here suggest that the chemisorption of dissolved O2 at buried Pt nanoparticle surfaces controls purification in the top-down case while both the high relative solubility coupled with weak physisorption of H2O explains the bottom-up process. Crucial too is the role that the carbonaceous contaminant itself has on the dissolution and diffusion of O2 and H2O. These results pave the way for simulation driven experiments where (1) the transient densification of the deposit can be accounted for in the initial deposit design stage and (2) the deposition and purification steps can be combined. PMID:26058775

  13. Electron nanoprobe induced oxidation: A simulation of direct-write purification

    DOE PAGESBeta

    Fowlkes, J. D.; Geier, B.; Lewis, B. B.; Rack, P. D.; Stanford, M. G.; Winkler, R.; Plank, H.

    2015-06-01

    Electron beam direct-write has recently taken a large step forward with the advent of methods to purify deposits. This development has opened the door for future direct-write device prototyping and editing. In one such approach, an additional beam scanning procedure removes carbonaceous impurities via oxidation from metal–carbon deposits (e.g., PtC5) in the presence of H2O or O2 after deposition. So far, critical aspects of the oxidation reaction remain unclear; experiments reveal clearly that electron stimulated oxidation drives the process yet it is not understood why H2O purifies by a bottom-up mechanism while O2 purifies from the top-down. The simulation resultsmore » presented here suggest that the chemisorption of dissolved O2 at buried Pt nanoparticle surfaces controls purification in the top-down case while both the high relative solubility coupled with weak physisorption of H2O explains the bottom-up process. Crucial too is the role that the carbonaceous contaminant itself has on the dissolution and diffusion of O2 and H2O. The results pave the way for simulation driven experiments where (1) the transient densification of the deposit can be accounted for in the initial deposit design stage and (2) the deposition and purification steps can be combined.« less

  14. Electron nanoprobe induced oxidation: A simulation of direct-write purification

    SciTech Connect

    Fowlkes, J. D.; Geier, B.; Lewis, B. B.; Rack, P. D.; Stanford, M. G.; Winkler, R.; Plank, H.

    2015-06-01

    Electron beam direct-write has recently taken a large step forward with the advent of methods to purify deposits. This development has opened the door for future direct-write device prototyping and editing. In one such approach, an additional beam scanning procedure removes carbonaceous impurities via oxidation from metal–carbon deposits (e.g., PtC5) in the presence of H2O or O2 after deposition. So far, critical aspects of the oxidation reaction remain unclear; experiments reveal clearly that electron stimulated oxidation drives the process yet it is not understood why H2O purifies by a bottom-up mechanism while O2 purifies from the top-down. The simulation results presented here suggest that the chemisorption of dissolved O2 at buried Pt nanoparticle surfaces controls purification in the top-down case while both the high relative solubility coupled with weak physisorption of H2O explains the bottom-up process. Crucial too is the role that the carbonaceous contaminant itself has on the dissolution and diffusion of O2 and H2O. The results pave the way for simulation driven experiments where (1) the transient densification of the deposit can be accounted for in the initial deposit design stage and (2) the deposition and purification steps can be combined.

  15. Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins.

    PubMed

    Béhar, Ghislaine; Renodon-Cornière, Axelle; Mouratou, Barbara; Pecorari, Frédéric

    2016-04-01

    Affinity chromatography is a convenient way of purifying proteins, as a high degree of purity can be reached in one step. The use of tags has greatly contributed to the popularity of this technique. However, the addition of tags may not be desirable or possible for the production of biopharmaceuticals. There is thus a need for tailored artificial affinity ligands. We have developed the use of archaeal extremophilic proteins as scaffolds to generate affinity proteins (Affitins). Here, we explored the potential of Affitins as ligand to design affinity columns. Affitins specific for human immunoglobulin G (hIgG), bacterial PulD protein, and chicken egg lysozyme were immobilized on an agarose matrix. The columns obtained were functional and highly selective for their cognate target, even in the presence of exogenous proteins as found in cell culture media, ascites and bacterial lysates, which result in a high degree of purity (∼95%) and recovery (∼100%) in a single step. Anti-hIgG Affitin columns withstand repetitive cycles of purification and cleaning-in-place treatments with 0.25 M NaOH as well as Protein A does. High levels of Affitin productions in Escherichia coli makes it possible to produce these affinity columns at low cost. Our results validate Affitins as a new class of tailored ligands for the affinity chromatography purification of potentially any proteins of interest including biopharmaceuticals. PMID:26952369

  16. Purification of native and recombinant cobra venom factor using thiophilic adsorption chromatography.

    PubMed

    Kölln, Johanna; Braren, Ingke; Bredehorst, Reinhard; Spillner, Edzard

    2007-01-01

    The complement activating venom component Cobra Venom Factor (CVF) forms a stable CVF-dependent C3 convertase complex, which initiates continuous activation of the complement system, consumes all downstream complement components and obliterates functional complement. Therefore, native CVF is routinely used as decomplementing agent in vivo and in vitro. However, in most countries, CVF and even unfractionated cobra venom are now becoming unavailable due to the CITES agreement. Although CVF is a complex molecule with three disulfide linked polypeptide chains and pronounced glycosylation, recombinant expression of the active molecule in eukaryotic host cells may provide an alternative source. In this study we describe a strategy for the production and efficient isolation of recombinant CVF from supernatant of mammalian cells. Thiophilic adsorption chromatography (TAC), an efficient procedure for purification of the human homologue C3, was evaluated for its suitability regarding purification of both native as well as recombinant CVF. Native CVF could be purified by TAC in a one-step procedure from cobra venom with yields of 92% compared to 35% by conventional approaches. After establishment of stably transfected mammalian cells recombinant CVF could be obtained and enriched from CHO supernatants by TAC to a purity of 73%, and up to 90% if an additional affinity chromatography step was included. Subsequent characterization revealed comparable hemolytic and bystander lysis activity and of rCVF and nCVF. These data demonstrate that the functional expression in mammalian cells in combination with TAC for purification renders rCVF a highly attractive substitute for its native counterpart. PMID:17584174

  17. Optimization of protein purification and characterization using Thermofluor screens.

    PubMed

    Boivin, Stephane; Kozak, Sandra; Meijers, Rob

    2013-10-01

    The efficient large scale production of recombinant proteins depends on the careful conditioning of the protein as it is isolated and purified to homogeneity. Low protein stability leads to low purification yields as a result of protein degradation, precipitation and folding instability. It is often necessary to go through several iterations of trial-and-error to optimize the homogeneity, stability and solubility of the protein sample. We have set up Thermofluor assays to identify customized protocols for the preparation and characterization of individual protein constructs. We apply a two-step approach: we first screen for global parameters, followed by a search for protein-specific additives. The first screen has been designed in such a way, that it is possible to discern global stability trends according to pH, salt concentration, buffer type and concentration. The second screen contains small molecules that can affect the folding, aggregation state and solubility of the protein construct and also includes small molecules that specifically bind and stabilize proteins. The screens are designed to evaluate purification and storage protocols, and aim to provide hints to optimize these protocols. The home-made screens have been tested on more than 200 different protein constructs at the Sample Preparation and Characterization (SPC) facility at EMBL Hamburg. We describe which RT-PCR machines can be adapted to perform Thermofluor assays, what are the necessary experimental conditions to set up a screen, some leads on how to interpret the data and we give several examples of Thermofluor applications beyond stability screens. PMID:23948764

  18. Stabilization and purification of tyrosine aminotransferase from rat liver.

    PubMed

    Hargrove, J L

    1990-01-01

    Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation by prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-glutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65 degrees C) followed by precipitation from 0.20 M sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6.0. These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity. PMID:1973296

  19. Purification and concentration of DNA from aqueous solutions.

    PubMed

    Moore, David; Dowhan, Dennis

    2002-08-01

    This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low-molecular-weight oligonucleotides and triphosphates. PMID:18265306

  20. Purification and concentration of DNA from aqueous solutions.

    PubMed

    Moore, D

    2001-05-01

    This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. The Basic Protocol, using phenol extraction and ethanol precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations <1 mg/ml. Isopropanol may also be used to precipitate DNA, as described in an alternate protocol. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads, and is described here. These protocol may also be used for purifying RNA. The final protocols provide modifications to the Basic Protocol that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing ow-molecular-weight oligonucleotides and triphosphates. PMID:18432672

  1. Purification and concentration of DNA from aqueous solutions.

    PubMed

    Moore, David; Dowhan, Dennis

    2007-09-01

    This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The Basic Protocol, using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether, respectively. An alternative to these methods is nucleic acid purification using glass beads, and this technique is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low-molecular-weight oligonucleotides and triphosphates. PMID:21948158

  2. Multiphoton-state-assisted entanglement purification of material qubits

    NASA Astrophysics Data System (ADS)

    Bernád, József Zsolt; Torres, Juan Mauricio; Kunz, Ludwig; Alber, Gernot

    2016-03-01

    We propose an entanglement purification scheme based on material qubits and ancillary coherent multiphoton states. We consider a typical QED scenario where material qubits implemented by two-level atoms fly sequentially through a cavity and interact resonantly with a single mode of the radiation field. We explore the theoretical possibilities of realizing a high-fidelity two-qubit quantum operation necessary for the purification protocol with the help of a postselective balanced homodyne photodetection. We demonstrate that the obtained probabilistic quantum operation can be used as a bilateral operation in the proposed purification scheme. It is shown that the probabilistic nature of this quantum operation is counterbalanced in the last step of the scheme where qubits are not discarded after inadequate qubit measurements. As this protocol requires present-day experimental setups and generates high-fidelity entangled pairs with high repetition rates, it may offer interesting perspectives for applications in quantum information theory.

  3. Isolation and Purification of Leaf Starch Components

    PubMed Central

    Chang, Chong W.

    1979-01-01

    A procedure was developed for the separation and purification of amylose and amylopectin isolated from cotton leaves. Cotton leaves were homogenized in 0.02 molar phosphate buffer at pH 7.0 containing HgCl2 plus toluene. Crude starch granules were collected by centrifugation and partially purified by treating with acetone and toluene. The starch granules were then dispersed in dimethylsulfoxide and precipitated with ethyl alcohol. The precipitate was suspended in boiling water. Amylose was separated from amylopectin and cell wall particles on a Sepharose 2B column and further purified with thymol and butanol. Amylopectin was then separated from the colloidal cell wall contaminants by its specific interaction with concanavalin A. Purities of starch components were verified by specific biochemical and enzymic tests in addition to their iodine-binding capacity. This procedure should also be suitable for purification of starch components from other plant sources. PMID:16661064

  4. Improved native affinity purification of RNA.

    PubMed

    Batey, Robert T; Kieft, Jeffrey S

    2007-08-01

    RNA biochemical or structural studies often require an RNA sample that is chemically pure, and most protocols for its in vitro production use denaturing polyacrylamide gel electrophoresis to achieve this. Unfortunately, many RNAs do not quantitatively refold into an active conformation after denaturation, creating significant problems for downstream characterization or use. In addition, this traditional purification method is not amenable to studies demanding high-throughput RNA production. Recently, we presented the first general method for producing almost any RNA sequence that employs an affinity tag that is removed during the purification process. Because technical difficulties prevented application of this method to many RNAs, we have developed an improved version that utilizes a different activatable ribozyme and affinity tag that are considerably more robust, rapid, and broadly applicable. PMID:17548432

  5. Oxygen Sag and Stream Purification.

    ERIC Educational Resources Information Center

    Neal, Larry; Herwig, Roy

    1978-01-01

    Presents a literature review of water quality related to oxygen sag and stream purification, covering publications of 1976-77. This review includes: (1) self-purification models; (2) oxygen demand; and (3) reaeration and oxygen transfer. A list of 60 references is also presented. (HM)

  6. DNA PURIFICATION BY POLYCARBONATE FILTERS

    EPA Science Inventory

    Organic solvent free procedure s are described for the purification of mammalian DNA from rat liver, kidney, spleen, lung and brain. he basis of the purification procedures are the use of the detergent sodium dodecyl sulfate (SDS) and inert polycarbonate filters with 2 um pores w...

  7. Magnetic purification of curcumin from Curcuma longa rhizome by novel naked maghemite nanoparticles.

    PubMed

    Magro, Massimiliano; Campos, Rene; Baratella, Davide; Ferreira, Maria Izabela; Bonaiuto, Emanuela; Corraducci, Vittorino; Uliana, Maíra Rodrigues; Lima, Giuseppina Pace Pereira; Santagata, Silvia; Sambo, Paolo; Vianello, Fabio

    2015-01-28

    Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids. PMID:25584520

  8. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes

    PubMed Central

    Kaiser, Peter; Meierhofer, David; Wang, Xiaorong; Huang, Lan

    2011-01-01

    Most biological processes are governed by multiprotein complexes rather than individual proteins. Identification of protein complexes therefore is becoming increasingly important to gain a molecular understanding of cells and organisms. Mass spectrometry–based proteomics combined with affinity-tag-based protein purification is one of the most effective strategies to isolate and identify protein complexes. The development of tandem-affinity purification approaches has revolutionized proteomics experiments. These two-step affinity purification strategies allow rapid, effective purification of protein complexes and, at the same time, minimize background. Identification of even very low-abundant protein complexes with modern sensitive mass spectrometers has become routine. Here, we describe two general strategies for tandem-affinity purification followed by mass spectrometric identification of protein complexes. PMID:18370112

  9. Production, Purification, and Capsid Stability of Rhinovirus C Types

    PubMed Central

    Griggs, Theodor F.; Bochkov, Yury A.; Nakagome, Kazuyuki; Palmenberg, Ann C.; Gern, James E.

    2015-01-01

    The Rhinovirus C (RV-C) were discovered in 2006 and these agents are an important cause of respiratory morbidity. Little is known about their biology. RV-C15 (C15) can be produced by transfection of recombinant viral RNA into cells and subsequent purification over a 30% sucrose cushion, even though yields and infectivity of other RV-C genotypes with this protocol are low. The goal of this study was to determine whether poor RV-C yields were due to capsid instability, and moreover, to develop a robust protocol suitable for the purification of many RV-C types. Capsid stability assays indicated that virions of RV-C41 (refractory to purification) have similar tolerance for osmotic and temperature stress as RV-A16 (purified readily), although C41 is more sensitive to low pH. Modification to the purification protocol by removing detergent increased the yield of RV-C. Addition of nonfat dry milk to the sucrose cushion increased the virus yield but sacrificed purity of the viral suspension. Analysis of virus distribution following centrifugation indicated that the majority of detectable viral RNA (vRNA) was found in pellets refractory to resuspension. Reduction of the centrifugal force with commiserate increase in spin-time improved the recovery of RV-C for both C41 and C2. Transfection of primary lung fibroblasts (WisL cells) followed by the modified purification protocol further improved yields of infectious C41 and C2. Described herein is a higher-yield purification protocol suitable for RV-C types refractory to the standard purification procedure. The findings suggest that aggregation-adhesion problems rather than capsid instability influence RV-C yield during purification. PMID:25724434

  10. Identification of protein interacting partners using tandem affinity purification.

    PubMed

    Bailey, Dalan; Urena, Luis; Thorne, Lucy; Goodfellow, Ian

    2012-01-01

    A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification(1). Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast(2,3) but more recently has been adapted to use in mammalian cells(4-8). As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E(9,10).The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation(10). The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence(8). To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter. Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous

  11. Water Purification Product

    NASA Technical Reports Server (NTRS)

    2004-01-01

    Ecomaster, an affiliate of BioServe Space Technologies, this PentaPure technology has been used to purify water for our nation's Space Shuttle missions since 1981. WTC-Ecomaster of Mirneapolis, Minnesota manufactures water purification systems under the brand name PentaPure (TM). BioServe researcher Dr. George Marchin, of Kansas State University, first demonstrated the superiority of this technology and licensed it to WTC. Marchin continues to perform microgravity research in the development of new technologies for the benefit of life on Earth.

  12. Air/Water Purification

    NASA Technical Reports Server (NTRS)

    1992-01-01

    After 18 years of research into air/water pollution at Stennis Space Center, Dr. B. C. Wolverton formed his own company, Wolverton Environmental Services, Inc., to provide technology and consultation in air and water treatment. Common houseplants are used to absorb potentially harmful materials from bathrooms and kitchens. The plants are fertilized, air is purified, and wastewater is converted to clean water. More than 100 U.S. communities have adopted Wolverton's earlier water hyacinth and artificial marsh applications. Catfish farmers are currently evaluating the artificial marsh technology as a purification system.

  13. Iterative Purification and Effect Size Use with Logistic Regression for Differential Item Functioning Detection

    ERIC Educational Resources Information Center

    French, Brian F.; Maller, Susan J.

    2007-01-01

    Two unresolved implementation issues with logistic regression (LR) for differential item functioning (DIF) detection include ability purification and effect size use. Purification is suggested to control inaccuracies in DIF detection as a result of DIF items in the ability estimate. Additionally, effect size use may be beneficial in controlling…

  14. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins.

    PubMed

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh

    2016-10-01

    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  15. Production and purification of Clostridium botulinum type C and D neurotoxin.

    PubMed

    Gessler, F; Böhnel, H

    1999-07-01

    Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well. A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure. Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up. The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs. Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively. A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay. PMID:10397323

  16. Purification of boron nitride nanotubes via polymer wrapping

    SciTech Connect

    Choi, Jin-Hyuk; Kim, Jaewoo; Seo, Duckbong; Seo, Young-Soo

    2013-03-15

    Highlights: ► Surface modification of boron nitride nanotubes using polymeric materials. ► Surface-modified BNNT was purified with a simple dilution-centrifugation step. ► Surface-modified BNNT can be directly used for polymer composite fabrication ► Degree of purification was analyzed by Raman spectroscopy. - Abstract: Boron nitride nanotubes (BNNT) synthesized by a ball milling-annealing were surface-modified using three different types of polymeric materials. Those materials were chosen depending on future applications especially in polymer nanocomposite fabrications. We found that the surface-modified BNNT can be purified with a simple dilution-centrifugation step, which would be suitable for large-scale purification. Degree of purification was monitored by means of the center peak position and FWHM of E{sub 2g} mode of BNNT in Raman spectra. As the purification of BNNT develops, the peak position was up-shifted while FWHM of the peak was narrowed.

  17. Stepped nozzle

    DOEpatents

    Sutton, G.P.

    1998-07-14

    An insert is described which allows a supersonic nozzle of a rocket propulsion system to operate at two or more different nozzle area ratios. This provides an improved vehicle flight performance or increased payload. The insert has significant advantages over existing devices for increasing nozzle area ratios. The insert is temporarily fastened by a simple retaining mechanism to the aft end of the diverging segment of the nozzle and provides for a multi-step variation of nozzle area ratio. When mounted in place, the insert provides the nozzle with a low nozzle area ratio. During flight, the retaining mechanism is released and the insert ejected thereby providing a high nozzle area ratio in the diverging nozzle segment. 5 figs.

  18. Stepped nozzle

    DOEpatents

    Sutton, George P.

    1998-01-01

    An insert which allows a supersonic nozzle of a rocket propulsion system to operate at two or more different nozzle area ratios. This provides an improved vehicle flight performance or increased payload. The insert has significant advantages over existing devices for increasing nozzle area ratios. The insert is temporarily fastened by a simple retaining mechanism to the aft end of the diverging segment of the nozzle and provides for a multi-step variation of nozzle area ratio. When mounted in place, the insert provides the nozzle with a low nozzle area ratio. During flight, the retaining mechanism is released and the insert ejected thereby providing a high nozzle area ratio in the diverging nozzle segment.

  19. Highly efficient Bell state purification and GHZ preparation and purification

    NASA Astrophysics Data System (ADS)

    Krastanov, Stefan; Jiang, Liang

    2016-05-01

    We investigate novel protocols for entanglement purification with Bell states. Employing genetic algorithms for the design of the purification circuit, we obtain shorter circuits giving higher success rates and better final fidelities than what is available in the literature. We generalize these circuits in order to prepare GHZ states from Bell pairs and to subsequently purify these GHZ states. We provide new threshold estimates for codes using these GHZ states for fault-tolerant stabilizer measurements.

  20. Acrylic purification and coatings

    SciTech Connect

    Kuzniak, Marcin

    2011-04-27

    Radon (Rn) and its decay daughters are a well-known source of background in direct WIMP detection experiments, as either a Rn decay daughter or an alpha particle emitted from a thin inner surface layer of a detector could produce a WIMP-like signal. Different surface treatment and cleaning techniques have been employed in the past to remove this type of contamination. A new method of dealing with the problem has been proposed and used for a prototype acrylic DEAP-1 detector. Inner surfaces of the detector were coated with a layer of ultra pure acrylic, meant to shield the active volume from alphas and recoiling nuclei. An acrylic purification technique and two coating techniques are described: a solvent-borne (tested on DEAP-1) and solvent-less (being developed for the full scale DEAP-3600 detector).

  1. Exhaust purification apparatus

    SciTech Connect

    Shinzawa, M.; Ushimura, S.

    1987-05-05

    An exhaust purification apparatus is described for use in an internal combustion engine having an exhaust conduit through which exhaust particles are discharged together with exhaust gas to the atmosphere. Included is an outer shell having an inlet connected to the exhaust conduit and an outlet connected to the atmosphere. The outer shell contains a trap element and a regenerative burner located upstream of the trap element, the regenerative burner comprising: a cylindrical hollow member fixed to the liner and extending within a combustion chamber to define an evaporation chamber, a glow plug for igniting the mixture supplied into the evaporated chamber when actuated; and a control unit responsive to a regeneration requirement for actuating the glow plug and supplying an air-fuel mixture into the evaporation chamber through the mixture conduit.

  2. URANIUM PURIFICATION PROCESS

    DOEpatents

    Ruhoff, J.R.; Winters, C.E.

    1957-11-12

    A process is described for the purification of uranyl nitrate by an extraction process. A solution is formed consisting of uranyl nitrate, together with the associated impurities arising from the HNO/sub 3/ leaching of the ore, in an organic solvent such as ether. If this were back extracted with water to remove the impurities, large quantities of uranyl nitrate will also be extracted and lost. To prevent this, the impure organic solution is extracted with small amounts of saturated aqueous solutions of uranyl nitrate thereby effectively accomplishing the removal of impurities while not allowing any further extraction of the uranyl nitrate from the organic solvent. After the impurities have been removed, the uranium values are extracted with large quantities of water.

  3. Purification of recombinant poly(ADP-ribose) polymerases.

    PubMed

    Amé, Jean-Christophe; Kalisch, Thomas; Dantzer, Françoise; Schreiber, Valérie

    2011-01-01

    The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture. PMID:21870259

  4. Recovery and purification of ethylene

    DOEpatents

    Reyneke, Rian; Foral, Michael J.; Lee, Guang-Chung; Eng, Wayne W. Y.; Sinclair, Iain; Lodgson, Jeffery S.

    2008-10-21

    A process for the recovery and purification of ethylene and optionally propylene from a stream containing lighter and heavier components that employs an ethylene distributor column and a partially thermally coupled distributed distillation system.

  5. A fast and easy strategy for protein purification using "teabags".

    PubMed

    Castaldo, M; Barlind, L; Mauritzson, F; Wan, P T; Snijder, H J

    2016-01-01

    Protein purification often involves affinity capture of proteins on stationary resin, alternatively proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment. PMID:27356497

  6. An efficient purification system for native minichromosome from S. cerevisiae

    PubMed Central

    Unnikrishnan, Ashwin; Akiyoshi, Bungo; Biggins, Sue; Tsukiyama, Toshio

    2011-01-01

    We have recently established a system for purifying minichromosome in a native state from S. cerevisiae. This system is extremely efficient, and a single-step purification yields samples with sufficient purity and quantity for mass spectrometry (MS) analysis of histones and non-histone proteins tightly associated with the minichromosome. The templates can also be used in various biochemical assays in vitro, such as transcription and recombination, and might be applicable to allow EM or other studies to be performed. PMID:22183591

  7. Production and Purification of Streptokinase by Protected Affinity Chromatography

    PubMed Central

    Babashamsi, Mohammad; Razavian, Mohammad Hossein; Nejadmoghaddam, Mohammad Reza

    2009-01-01

    Streptokinase is an extracellular protein, extracted from certain strains of beta hemolytic streptococcus. It is a non-protease plasminogen activator that activates plasminogen to plasmin, the enzyme that degrades fibrin cloth through its specific lysine binding site; it is used therefore as a drug in thrombolytic therapy. The rate of bacterial growth and streptokinase production was studied in condition of excess glucose addition to culture media and its pH maintenance. The streptokinase product of the bacterial culture was preliminary extracted by salt precipitation and then purified by affinity chromatography on plasminogen substituted sepharose-4B in a condition that the plasminogen active site was protected from streptokinase-induced activation. The purity of streptokinase was confirmed by SDS-PAGE and its biological activity determined in a specific streptokinase assay. The results showed that in the fed–batch culture, the rate of streptokinase production increased over two times as compared with the batch culture while at the same time, shortening the streptokinase purification to a single step increased the yield over 95% at the chromatography stage. PMID:23407807

  8. Purification of Δ(5)-3-ketosteroid isomerase from Digitalis lanata.

    PubMed

    Meitinger, Nadine; Geiger, Daniel; Augusto, Thierry W; Maia de Pádua, Rodrigo; Kreis, Wolfgang

    2015-01-01

    The isomerization of 5-pregnene-3,20-dione into 4-pregnene-3,20-dione was investigated to shed further light on cardenolide biosynthesis and to characterize the enzymes involved in cardenolide formation. It was shown that the Δ(5)-3-ketosteroid isomerase of Digitalis lanata, which catalyzes this isomerization, is an individual enzyme and not, as previously thought, associated with Δ(5)-3β-hydroxysteroid dehydrogenase. The enzyme was purified by fractionated ammonium sulfate precipitation, hydrophobic interaction chromatography and gel filtration. The purification protocol resulted in a 68.1-fold enriched specific enzyme activity with a yield of 2.2%. After an additional chromatofocusing step the 3KSI activity appeared as a single protein band at 17kDa in SDS-PAGE. Plant 3KSI displayed similar properties to microbial 3-ketosteroid isomerases. PMID:25468533

  9. Purification and concentration of alkaline phosphatase by selective permeabilization of Escherichia coli using reverse micellar solutions.

    PubMed

    Bansal-Mutalik, Ritu; Gaikar, Vilas G

    2003-01-01

    Recovery of alkaline phosphatase (AP) from the periplasm of Escherichia coli using reverse micellar solutions (RMSs) of sodium dioctyl sulfosuccinate (AOT) in aliphatic hydrocarbons has been attempted. A variety of surface-active agents, solvents, and reverse micellar conditions were screened, and an excellent recovery of the enzyme in a concentrated form, with a high purification factor, was obtained in a single-step process. The permeabilization process strongly depended on the water content of the RMS as well as on the amount of water coating the microbial cell surface. The product was almost free from nucleic acids. In addition, because of the low affinity of AOT and the organic solvent for the aqueous phase, contamination by the permeabilizing agents would also be negligible. PMID:14656146

  10. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens.

    PubMed

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies. PMID:26713728

  11. Purification and Characterization of Recombinant Human Lysozyme from Eggs of Transgenic Chickens

    PubMed Central

    Wu, Hanyu; Cao, Dainan; Liu, Tongxin; Zhao, Jianmin; Hu, Xiaoxiang; Li, Ning

    2015-01-01

    Transgenic chickens as bioreactors have several advantages, such as the simple establishment procedure, correct glycosylation profile of expressed proteins, etc. Lysozyme is widely used in food industry, livestock farming, and medical field as a replacement of antibiotics because of its antibacterial and complement system-modulating activity. In this study, we used RT-PCR, Western blot, and immunofluorescence to detect the expression of recombinant human lysozyme (rhLY) in the transgenic chicken. We demonstrated that the transgene of rhLY was genetically stable across different generations. We next optimized the purification procedure of rhLY from the transgenic eggs by utilizing two steps of cation-exchange chromatography and one gel-filtration chromatography. About 6 mg rhLY with the purity exceeding 90% was obtained from ten eggs, and the purification efficiency was about 75%. The purified rhLY had similar physicochemical and biological properties in molecular mass and antibacterial activity compared to the commercial human lysozyme. Additionally, both of them exhibited thermal stability at 60°C and tolerated an extensive pH range of 2 to 11. In conclusion, our study proved that the transgenic chickens we have previously generated were genetically stable and suitable for the production of active rhLY. We also provided a pipeline for purifying the recombinant proteins from transgenic eggs, which could be useful for other studies. PMID:26713728

  12. Purification and characterization of the Oligosaccharyl transferase

    SciTech Connect

    Kapoor, T.M.

    1990-11-01

    Oligosaccharyl transferase was characterized to be a glycoprotein with at least one saccharide unit that had a D-manno or D- glucopyranose configuration with unmodified hydroxy groups at C-3, C-4 and C-6, using a Concanavalin A affinity column. This afforded a 100 fold increase in the transferase purity in the solubilized microsomal sample and also removed over 90% of the microsomal proteins (the cytosolic ones being removed before solubilization). The detergent, N,N-Dimethyldodecylamine N-oxide (LDAO) was used for solubilization and it yielded a system compatible with the assay and the purification steps. An efficient method for detergent extraction without dilution of sample or protein precipitation was also developed.

  13. Purification of uranothorite solid solutions from polyphase systems

    NASA Astrophysics Data System (ADS)

    Clavier, Nicolas; Szenknect, Stéphanie; Costin, Dan Tiberiu; Mesbah, Adel; Ravaux, Johann; Poinssot, Christophe; Dacheux, Nicolas

    2013-10-01

    The mineral coffinite, nominally USiO4, and associated Th1-xUxSiO4 uranothorite solid solutions are of great interest from a geochemical point of view and in the case of the direct storage of spent nuclear fuels. Nevertheless, they clearly exhibit a lack in the evaluation of their thermodynamic data, mainly because of the difficulties linked with their preparation as pure phases. This paper thus presents physical and chemical methods aiming to separate uranothorite solid solutions from oxide additional phases such as amorphous SiO2 and nanometric crystallized Th1-yUyO2. The repetition of centrifugation steps envisaged in first place was rapidly dropped due to poor recovery yields, to the benefit of successive washings in acid then basic media. Under both static and dynamic flow rates (i.e. low or high rate of leachate renewal), ICP-AES (Inductively Coupled Plasma - Atomic Emission Spectroscopy) analyses revealed the systematic elimination of Th1-yUyO2 in acid media and of SiO2 in basic media. Nevertheless, two successive steps were always needed to reach pure samples. On this basis, a first cycle performed in static conditions was chosen to eliminate the major part of the accessory phases while a second one, in dynamic conditions, allowed the elimination of the residual impurities. The complete purification of the samples was finally evidenced through the characterization of the samples by the means of PXRD (Powder X-Ray Diffraction), SEM (Scanning Electron Microscopy) observations and X-EDS (X-Ray Energy Dispersive Spectroscopy) analyses.

  14. Purification of a rat neurotensin receptor expressed in Escherichia coli.

    PubMed Central

    Tucker, J; Grisshammer, R

    1996-01-01

    A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress. PMID:8760379

  15. Purification of gibberellin sub 53 -oxidase from spinach

    SciTech Connect

    Wilson, T.M.; Zeevaart, J.A.D. )

    1989-04-01

    Spinach is a long-day rosette plants, in which stem growth is mediated by gibberellins. It has been shown that two enzymatic steps, GA{sub 53}-oxidase and GA{sub 19}-oxidase, are controlled by light. To develop an understanding into this light regulation, purification of GA{sub 53}-oxidase has been undertaken. The original assay relied on the HPLC separation of the product and substrate, but was considered too slow for the development of a purification scheme. A TLC system was developed which in conjunction with improvements to the assay conditions was sensitive and gave rapid results. The partial purification of the GA{sub 53}-oxidase is achieved by a high speed centrifugation, 40-55% ammonium sulfate precipitation, an hydroxyapatite column, Sephadex G-100 column and an anion exchange FPLC column, Mono Q HR10/10, yielding 1000-fold purification and 15% recovery. Monoclonal antibodies to the protein will be raised and used to further characterize the enzyme.

  16. Water Purification Systems

    NASA Technical Reports Server (NTRS)

    1994-01-01

    Clearwater Pool Technologies employs NASA-developed silver/copper ionization to purify turtle and dolphin tanks, cooling towers, spas, water recycling systems, etc. The pool purifier consists of a microcomputer to monitor water conditions, a pair of metallic electrodes, and a rheostat controller. Ions are generated by passing a low voltage current through the electrodes; the silver ions kill the bacteria, and the copper ions kill algae. This technology has found broad application because it offers an alternative to chemical disinfectants. It was originally developed to purify water on Apollo spacecraft. Caribbean Clear has been using NASA's silver ionization technology for water purification for more than a decade. Two new products incorporate advancements of the basic technology. One is the AquaKing, a system designed for areas with no source of acceptable drinking water. Another is the Caribbean Clear Controller, designed for commercial pool and water park applications where sanitizing is combined with feedback control of pH and an oxidizer, chlorine or bromine. The technology was originally developed to purify water on Apollo spacecraft.

  17. Purification of recombinant protein by cold-coacervation of fusion constructs incorporating resilin-inspired polypeptides.

    PubMed

    Lyons, Russell E; Elvin, Christopher M; Taylor, Karin; Lekieffre, Nicolas; Ramshaw, John A M

    2012-12-01

    Polypeptides containing between 4 and 32 repeats of a resilin-inspired sequence AQTPSSYGAP, derived from the mosquito Anopheles gambiae, have been used as tags on recombinant fusion proteins. These repeating polypeptides were inspired by the repeating structures that are found in resilins and sequence-related proteins from various insects. Unexpectedly, an aqueous solution of a recombinant resilin protein displays an upper critical solution temperature (cold-coacervation) when held on ice, leading to a separation into a protein rich phase, typically exceeding 200 mg/mL, and a protein-poor phase. We show that purification of recombinant proteins by cold-coacervation can be performed when engineered as a fusion partner to a resilin-inspired repeat sequence. In this study, we demonstrate the process by the recombinant expression and purification of enhanced Green fluorescent protein (EGFP) in E. coli. This facile purification system can produce high purity, concentrated protein solutions without the need for affinity chromatography or other time-consuming or expensive purification steps, and that it can be used with other bulk purification steps such as low concentration ammonium sulfate precipitation. Protein purification by cold-coacervation also minimizes the exposure of the target protein to enhanced proteolysis at higher temperature. PMID:22627880

  18. Preparation of group I introns for biochemical studies and crystallization assays by native affinity purification.

    PubMed

    Vicens, Quentin; Gooding, Anne R; Duarte, Luis F; Batey, Robert T

    2009-01-01

    The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides -- some containing exons -- were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3' overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules. PMID:19710925

  19. SPAR-H Step-by-Step Guidance

    SciTech Connect

    April M. Whaley; Dana L. Kelly; Ronald L. Boring; William J. Galyean

    2012-06-01

    Step-by-step guidance was developed recently at Idaho National Laboratory for the US Nuclear Regulatory Commission on the use of the Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) method for quantifying Human Failure Events (HFEs). This work was done to address SPAR-H user needs, specifically requests for additional guidance on the proper application of various aspects of the methodology. This paper overviews the steps of the SPAR-H analysis process and highlights some of the most important insights gained during the development of the step-by-step directions. This supplemental guidance for analysts is applicable when plant-specific information is available, and goes beyond the general guidance provided in existing SPAR-H documentation. The steps highlighted in this paper are: Step-1, Categorizing the HFE as Diagnosis and/or Action; Step-2, Rate the Performance Shaping Factors; Step-3, Calculate PSF-Modified HEP; Step-4, Accounting for Dependence, and; Step-5, Minimum Value Cutoff.

  20. Sticky steps inhibit step motions near equilibrium

    NASA Astrophysics Data System (ADS)

    Akutsu, Noriko

    2012-12-01

    Using a Monte Carlo method on a lattice model of a vicinal surface with a point-contact-type step-step attraction, we show that, at low temperature and near equilibrium, there is an inhibition of the motion of macrosteps. This inhibition leads to a pinning of steps without defects, adsorbates, or impurities (self-pinning of steps). We show that this inhibition of the macrostep motion is caused by faceted steps, which are macrosteps that have a smooth side surface. The faceted steps result from discontinuities in the anisotropic surface tension (the surface free energy per area). The discontinuities are brought into the surface tension by the point-contact-type step-step attraction. The point-contact-type step-step attraction also originates “step droplets,” which are locally merged steps, at higher temperatures. We derive an analytic equation of the surface stiffness tensor for the vicinal surface around the (001) surface. Using the surface stiffness tensor, we show that step droplets roughen the vicinal surface. Contrary to what we expected, the step droplets slow down the step velocity due to the diminishment of kinks in the merged steps (smoothing of the merged steps).

  1. Biochemistry of Vibrio cholerae virulence: purification of cholera enterotoxin by preparative disc electrophoresis.

    PubMed Central

    Lewis, A C; Richardson, S H; Sheridan, B

    1976-01-01

    Procedures for cholera enterotoxin purification previously developed in this labarotory were not applicable to large-scale purification, and these methods resulted in low yields of pure toxin. An efficient scheme has been developed whereby pure cholera enterotoxin can be obtained from 6 to 8 liters of culture supernatant fluid. This method consists of concentration by membrane ultrafiltration followed by gel filtration and cation-exchange chromatography. Pure cholera enterotoxin of high biological potency was obtained after a final step of preparative acrylamide gel electrophoresis. The degree of purity of the toxin-antigen as well as its biological activity were determined at various setps of purification. This alternate technique for purification is offered because of the widespread interest in cholera enterotoxin as a specific stimulator of adenyl cyclase. Images PMID:987751

  2. Argon Purification Reference and Recommendation

    SciTech Connect

    Wu, J.; /Fermilab

    1991-05-23

    This engineering note is a reference for future consideration on the purification of argon. The original concern was for the possibility of argon contamination from components in the cryostats over long-term storage. An argon purification system could also be useful for purifying the contents of the argon dewar. The general conclusion is that most of the systems researched are too expensive at this time, but the recommended choice would be Centorr Furnaces. There were three basic types of purification systems which were to be considered. The first was the molecular sieve. This method would have been the preferred one, because it was claimed that it could purify liquid argon, removing liquid oxygen from the argon. However, none of the commercial companies researched provided this type of purification for use with liquid argon. Most companies said that this type of purification was impossible, and tests at IB-4 confirmed this. The second system contained a copper oxide to remove gaseous oxygen from argon gas. The disadvantage of this system wass that the argon had to be heated to a gas, and then cooled back down to liquid. The third system was similar to the second, except that it used tungsten or another material like titanium. This system also needed to heat the argon to gas, however the advantage of this system was that it supposedly removed all contaminants, that is, everything except for inert gases. Of the three systems, the third is the type manufactured by Centorr Furnaces, which uses a titanium charge.

  3. Entanglement purification with double selection

    SciTech Connect

    Fujii, Keisuke; Yamamoto, Katsuji

    2009-10-15

    We investigate an entanglement purification protocol with double-selection process, which works under imperfect local operations. Compared with the usual protocol with single selection, this double-selection method has higher noise thresholds for the local operations and quantum communication channels and achieves higher fidelity of purified states. It also provides a yield comparable to that of the usual protocol with single selection. We discuss on general grounds how some of the errors which are introduced by local operations are left as intrinsically undetectable. The undetectable errors place a general upper bound on the purification fidelity. The double selection is a simple method to remove all the detectable errors in the first order, so that the upper bound on the fidelity is achieved in the low-noise regime. The double selection is further applied to purification of multipartite entanglement such as two-colorable graph states.

  4. Hydraulic Design of Stepped Spillways Workshop

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Stepped chutes and spillways are commonly used for routing discharges during flood events. In addition, stepped chutes are used for overtopping protection of earthen embankments. Stepped spillways provide significant energy dissipation due to its stepped feature; as a result, the stilling basin as...

  5. [Purification and characterization of a bromoperoxidase from Gracilaria lemaneiformis].

    PubMed

    Li, Haiyan; Jin, Yan; Zhang, Wei; Yu, Xingju; Zhang, Jinyou; Wu, Peichun

    2008-04-01

    A bromoperoxidase from Gracilaria lemaneiformis was purified to homogeneity using a multi-step process of ammonium sulfate precipitation (AS), dialysis, and DEAE-cellulose 52 anion exchange chromatography. The bromoperoxidase activity was unstable or undetectable in crude extract solution. However, it became stable with electrophoretic purity after this multiple purification process. The anion exchange chromatography purification was a critical step in the purification process, which effectively eliminated the phycobiliprotein and smucilaginous polysaccharides. The purified bromoperoxidase was a monomeric enzyme with the relative molecular masses of 66 kD as determined by denaturing and native gradient gel electrophoresis. The optimal pH for bromoination was 6.0 and bromoperoxidase activity was stable as stored at a broad pH range of 3.0-9.0. Of a range of compounds tested, only vanadium enhanced bromoperoxidase activity. Kinetic studies for the bromination of monochlorodimedone (MCD) showed that the Km values of Br- and H2O2 are 53.5 micromol/L, 38 micromol/L respectively. PMID:18616173

  6. Step-step interactions on GaAs (110) nanopatterns

    SciTech Connect

    Galiana, B.; Benedicto, M.; Tejedor, P.

    2013-01-14

    The step-step interactions on vicinal GaAs (110) surface patterns have been extracted from the quantitative analysis of the terrace width distribution (TWD). We have specifically studied the interactions in near-equilibrium faceting and kinetics-driven step bunching and meandering formed by spontaneous self-organization or through the modification of GaAs growth kinetics by atomic hydrogen. We show that the experimental TWDs determined from atomic force microscopy measurements can be accurately described by a weighed sum of a generalized Wigner distribution and several Gaussians. The results of our calculations indicate that straight facets are formed during high temperature homoepitaxy due to attractive interactions between [110] steps. At low temperatures, steady state attractive interactions in [110] step bunches are preceded by a transition regime dominated by entropic and energetic repulsions between meandering [11n]-type steps (n {>=} 2), whose population density exceeds that of the [110] bunched steps. In addition, it has been found that atomic H reduces the attractive interactions between [110] bunched steps and enhances entropic and dipole-induced energetic repulsions between H-terminated [11n] steps through the inhibition of As-As bond formation at step edges. Our analysis has evidenced a correlation between the value of the adjustable parameter that accounts in our model for the specific weight of the secondary peaks in the TWD ({beta}) and the extent of transverse meandering on the vicinal surface.

  7. Development of a chamber system for rapid, high yield and cost-effective purification of deoxyribonucleic acid fragments from agarose gel

    PubMed Central

    Eslami, Gilda; Salehi, Rasoul

    2014-01-01

    Background: There are several methods commonly practicing for deoxyribonucleic acid (DNA) purification from agarose gel. In most laboratories, especially in developing countries, present methods for recovering of DNA fragments from the gel are mostly involved organic solvents. However, manual purification using organic solvents are toxic, labor intensive, time consuming and prone to contamination owing to several handling steps. The above mentioned burdens as well as cost and long time to import them, especially in developing countries, prompted us to design and develop a chamber system for rapid, non-toxic, cost-effective and user friendly device for polymerase chain reaction (PCR) products purification from agarose gel. Materials and Methods: The device was made from plexiglass plates. After amplification of two fragments of 250 and 850 bp, PCR products were electrophoresed. Subsequently, the desired bands were excised and purified with three method: HiPer Mini chamber, phenol extraction method and spin column procedure. To assess the suitability of the purified DNAs, restriction digestion was applied. Results: Results showed that the yield of recovered DNA in our method was above 95%, whereas the yields obtained with conventional phenol extraction and spin column methods were around 60%. Conclusion: In conclusion, the current method for DNA elution is quick, inexpensive and robust and it does not require the use of toxic organic solvents. In addition, the purified DNA was well has suited for further manipulations such as restriction digestion, ligation, cloning, sequencing and hybridization. PMID:24761386

  8. Sequential purification and crystal growth for the production of low cost silicon substrates. Annual report, 15 September 1979-14 September 1980

    SciTech Connect

    Liaw, M; D'Aragona, F S

    1980-01-01

    The objective of this program is to identify and develop low cost processing for fabricating large grain size polycrystalline silicon substrates. Metallurgical grade silicon (MG-Si) which is low cost and abundant for industrial usage was chosen as starting material. However, MG-Si cannot be used directly as substrates for solar cell fabrication for the following reasons: (1) it contains 1 to 2% metallic impurities, and (2) it is produced as irregular shapes with a fine grain structure. Various purification techniques have been reported. The techniques being studied under this program use direct methods for the purification of MG-Si. The process uses sequential steps of purification followed by crystal growth. The steps of sequential purification include: (1) leaching of MG-Si charge, (2) phase separation of non-soluble impurities from molten silicon, (3) reactive gas treatment of molten silicon, (4) liquid-liquid extraction (called slagging), and (5) impurity redistribution using ingot pulling. All the purification steps, with the exception of step (1), are performed in a consecutive manner using a crystal puller. The purified ingots will be produced in a desired ingot dimension and further recrystallization is not necessary. The theory and experimental results for each purification technique are presented. The relative effectiveness of the various steps are assessed and the most important step(s) are recommended. Finally the electrical characteristics of solar cells built on a thin epitaxial layer deposited on single pulled MG-Si substrates are discussed and compared to single crystal substrates. (WHK)

  9. Purification of adenovirus hexon by high performance liquid chromatography.

    PubMed

    Siegel, S A; Hutchins, J E; Witt, D J

    1987-09-01

    Hexon is the major structural protein of adenovirus, and has significance in studies of virus structure and function, vaccine development, and immunodiagnosis. We describe a simple, single-step, anion-exchange high performance liquid chromatography (HPLC) method for the high yield purification of hexon. Purity of the isolated hexon was assessed by SDS-PAGE and HPLC methods. The isolated hexon was immunologically reactive with anti-hexon monoclonal antibody in a dot-blot assay. It also retained immunogenicity, as polyclonal antisera from rabbits immunized with hexon showed the desired antigen specificity. The enhanced speed of this purification method allows for the efficient isolation of hexon from various serotypes, and thus may facilitate comparative studies of hexon immunobiology. PMID:3680460

  10. Fast multi-copy entanglement purification with linear optics

    NASA Astrophysics Data System (ADS)

    Cai, Chun; Zhou, Lan; Sheng, Yu-Bo

    2015-12-01

    We describe an entanglement purification protocol for a polarization Bell state. Different from the previous protocols, it does not require the controlled-not gate, and only uses linear optical elements to complete the task. This protocol requires multi-copy degraded mixed states, which can make this protocol obtain a high fidelity in one purification step. It can also be extended to purify the multi-photon Greenberger-Horne-Zeilinger (GHZ) state. This protocol may be useful in future long-distance communication. Project supported by the National Natural Science Foundation of China (Grant Nos. 11474168 and 61401222), the Qing Lan Project of Jiangsu Province, China, the STITP Project in Nanjing University of Posts and Telecommunications, the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20151502), the Natural Science Foundation of the Jiangsu Higher Education Institutions (Grant No. 15KJA120002), and the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

  11. Native Purification and Analysis of Long RNAs

    PubMed Central

    Chillón, Isabel; Marcia, Marco; Legiewicz, Michal; Liu, Fei; Somarowthu, Srinivas; Pyle, Anna Marie

    2015-01-01

    The purification and analysis of long noncoding RNAs (lncRNAs) in vitro is a challenge, particularly if one wants to preserve elements of functional structure. Here, we describe a method for purifying lncRNAs that preserves the cotranscriptionally derived structure. The protocol avoids the misfolding that can occur during denaturation–renaturation protocols, thus facilitating the folding of long RNAs to a native-like state. This method is simple and does not require addition of tags to the RNA or the use of affinity columns. LncRNAs purified using this type of native purification protocol are amenable to biochemical and biophysical analysis. Here, we describe how to study lncRNA global compaction in the presence of divalent ions at equilibrium using sedimentation velocity analytical ultracentrifugation and analytical size-exclusion chromatography as well as how to use these uniform RNA species to determine robust lncRNA secondary structure maps by chemical probing techniques like selective 2′-hydroxyl acylation analyzed by primer extension and dimethyl sulfate probing. PMID:26068736

  12. HPLC analysis and purification of peptides.

    PubMed

    Mant, Colin T; Chen, Yuxin; Yan, Zhe; Popa, Traian V; Kovacs, James M; Mills, Janine B; Tripet, Brian P; Hodges, Robert S

    2007-01-01

    High-performance liquid chromatography (HPLC) has proved extremely versatile over the past 25 yr for the isolation and purification of peptides varying widely in their sources, quantity and complexity. This article covers the major modes of HPLC utilized for peptides (size-exclusion, ion-exchange, and reversed-phase), as well as demonstrating the potential of a novel mixed-mode hydrophilic interaction/cation-exchange approach developed in this laboratory. In addition to the value of these HPLC modes for peptide separations, the value of various HPLC techniques for structural characterization of peptides and proteins will be addressed, e.g., assessment of oligomerization state of peptides/proteins by size-exclusion chromatography and monitoring the hydrophilicity/hydrophobicity of amphipathic alpha-helical peptides, a vital precursor for the development of novel antimicrobial peptides. The value of capillary electrophoresis for peptide separations is also demonstrated. Preparative reversed-phase chromatography purification protocols for sample loads of up to 200 mg on analytical columns and instrumentation are introduced for both peptides and recombinant proteins. PMID:18604941

  13. Entanglement purification for quantum communication

    NASA Astrophysics Data System (ADS)

    Pan, Jian-Wei; Simon, Christoph; Brukner, Časlav; Zeilinger, Anton

    2001-04-01

    The distribution of entangled states between distant locations will be essential for the future large-scale realization of quantum communication schemes such as quantum cryptography and quantum teleportation. Because of unavoidable noise in the quantum communication channel, the entanglement between two particles is more and more degraded the further they propagate. Entanglement purification is thus essential to distil highly entangled states from less entangled ones. Existing general purification protocols are based on the quantum controlled-NOT (CNOT) or similar quantum logic operations, which are very difficult to implement experimentally. Present realizations of CNOT gates are much too imperfect to be useful for long-distance quantum communication. Here we present a scheme for the entanglement purification of general mixed entangled states, which achieves 50 per cent of the success probability of schemes based on the CNOT operation, but requires only simple linear optical elements. Because the perfection of such elements is very high, the local operations necessary for purification can be performed with the required precision. Our procedure is within the reach of current technology, and should significantly simplify the implementation of long-distance quantum communication.

  14. Entanglement purification for quantum communication.

    PubMed

    Pan, J W; Simon, C; Brukner, C; Zeilinger, A

    2001-04-26

    The distribution of entangled states between distant locations will be essential for the future large-scale realization of quantum communication schemes such as quantum cryptography and quantum teleportation. Because of unavoidable noise in the quantum communication channel, the entanglement between two particles is more and more degraded the further they propagate. Entanglement purification is thus essential to distil highly entangled states from less entangled ones. Existing general purification protocols are based on the quantum controlled-NOT (CNOT) or similar quantum logic operations, which are very difficult to implement experimentally. Present realizations of CNOT gates are much too imperfect to be useful for long-distance quantum communication. Here we present a scheme for the entanglement purification of general mixed entangled states, which achieves 50 per cent of the success probability of schemes based on the CNOT operation, but requires only simple linear optical elements. Because the perfection of such elements is very high, the local operations necessary for purification can be performed with the required precision. Our procedure is within the reach of current technology, and should significantly simplify the implementation of long-distance quantum communication. PMID:11323664

  15. Anion exchange purification of plasmid DNA using expanded bed adsorption.

    PubMed

    Ferreira, G N; Cabral, J M; Prazeres, D M

    2000-01-01

    Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH = 8.0) buffer at an upward flow of 300 cmh-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh-1. Purification factors of 36 +/- 1 fold, 26 +/- 0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed-values of 35 +/- 2 and 5 +/- 0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step. PMID:10840595

  16. Isolation and Purification of Biotechnological Products

    NASA Astrophysics Data System (ADS)

    Hubbuch, Jürgen; Kula, Maria-Regina

    2007-05-01

    The production of modern pharma proteins is one of the most rapid growing fields in biotechnology. The overall development and production is a complex task ranging from strain development and cultivation to the purification and formulation of the drug. Downstream processing, however, still accounts for the major part of production costs. This is mainly due to the high demands on purity and thus safety of the final product and results in processes with a sequence of typically more than 10 unit operations. Consequently, even if each process step would operate at near optimal yield, a very significant amount of product would be lost. The majority of unit operations applied in downstream processing have a long history in the field of chemical and process engineering; nevertheless, mathematical descriptions of the respective processes and the economical large-scale production of modern pharmaceutical products are hampered by the complexity of the biological feedstock, especially the high molecular weight and limited stability of proteins. In order to develop new operational steps as well as a successful overall process, it is thus a necessary prerequisite to develop a deeper understanding of the thermodynamics and physics behind the applied processes as well as the implications for the product.

  17. Genotyping of plant and animal samples without prior DNA purification.

    PubMed

    Chum, Pak Y; Haimes, Josh D; André, Chas P; Kuusisto, Pia K; Kelley, Melissa L

    2012-01-01

    The Direct PCR approach facilitates PCR amplification directly from small amounts of unpurified samples, and is demonstrated here for several plant and animal tissues (Figure 1). Direct PCR is based on specially engineered Thermo Scientific Phusion and Phire DNA Polymerases, which include a double-stranded DNA binding domain that gives them unique properties such as high tolerance of inhibitors. PCR-based target DNA detection has numerous applications in plant research, including plant genotype analysis and verification of transgenes. PCR from plant tissues traditionally involves an initial DNA isolation step, which may require expensive or toxic reagents. The process is time consuming and increases the risk of cross contamination. Conversely, by using Thermo Scientific Phire Plant Direct PCR Kit the target DNA can be easily detected, without prior DNA extraction. In the model demonstrated here, an example of derived cleaved amplified polymorphic sequence analysis (dCAPS) is performed directly from Arabidopsis plant leaves. dCAPS genotyping assays can be used to identify single nucleotide polymorphisms (SNPs) by SNP allele-specific restriction endonuclease digestion. Some plant samples tend to be more challenging when using Direct PCR methods as they contain components that interfere with PCR, such as phenolic compounds. In these cases, an additional step to remove the compounds is traditionally required. Here, this problem is overcome by using a quick and easy dilution protocol followed by Direct PCR amplification (Figure 1). Fifteen year-old oak leaves are used as a model for challenging plants as the specimen contains high amounts of phenolic compounds including tannins. Gene transfer into mice is broadly used to study the roles of genes in development, physiology and human disease. The use of these animals requires screening for the presence of the transgene, usually with PCR. Traditionally, this involves a time consuming DNA isolation step, during which DNA

  18. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants.

    PubMed

    Sainsbury, Frank; Jutras, Philippe V; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  19. Engineering a recyclable elastin-like polypeptide capturing scaffold for non-chromatographic protein purification.

    PubMed

    Liu, Fang; Chen, Wilfred

    2013-01-01

    Previously, we reported a non-chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin-like polypeptide (ELP) to provide fast and cost-effective protein purification. However, the bound dockerin-intein tag cannot be completely dissociated from the ELP-cohesin capturing scaffold due to the high binding affinity, resulting in a single-use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium-coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA-mediated dissociation of the bound dockerin-intein tag from the ELP-cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non-chromatographic based affinity method provides an attractive approach for efficient and cost-effective protein purification. PMID:23801586

  20. A Chimeric Affinity Tag for Efficient Expression and Chromatographic Purification of Heterologous Proteins from Plants

    PubMed Central

    Sainsbury, Frank; Jutras, Philippe V.; Vorster, Juan; Goulet, Marie-Claire; Michaud, Dominique

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study, we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizing fusion partner SlCYS8. We show that the Cysta-tag may be used to readily purify proteins under native conditions, and then be removed enzymatically to isolate the protein of interest. We also show that commonly used protease recognition sites for linking purification tags are differentially stable in leaves of the commonly used expression host Nicotiana benthamiana, with those linkers susceptible to cysteine proteases being less stable then serine protease-cleavable linkers. As an example, we describe a Cysta-tag experimental scheme for the one-step purification of a clinically useful protein, human α1-antitrypsin, transiently expressed in N. benthamiana. With potential applicability to the variety of chromatography formats commercially available for IMAC-based protein purification, the Cysta-tag provides a convenient means for the efficient and cost-effective purification of recombinant proteins from plant tissues. PMID:26913045

  1. 2D nanostructures for water purification: graphene and beyond.

    PubMed

    Dervin, Saoirse; Dionysiou, Dionysios D; Pillai, Suresh C

    2016-08-18

    Owing to their atomically thin structure, large surface area and mechanical strength, 2D nanoporous materials are considered to be suitable alternatives for existing desalination and water purification membrane materials. Recent progress in the development of nanoporous graphene based materials has generated enormous potential for water purification technologies. Progress in the development of nanoporous graphene and graphene oxide (GO) membranes, the mechanism of graphene molecular sieve action, structural design, hydrophilic nature, mechanical strength and antifouling properties and the principal challenges associated with nanopore generation are discussed in detail. Subsequently, the recent applications and performance of newly developed 2D materials such as 2D boron nitride (BN) nanosheets, graphyne, molybdenum disulfide (MoS2), tungsten chalcogenides (WS2) and titanium carbide (Ti3C2Tx) are highlighted. In addition, the challenges affecting 2D nanostructures for water purification are highlighted and their applications in the water purification industry are discussed. Though only a few 2D materials have been explored so far for water treatment applications, this emerging field of research is set to attract a great deal of attention in the near future. PMID:27506268

  2. Synthesis and purification of 1,3,5-triamino-2,4,6-trinitrobenzene (TATB)

    DOEpatents

    Mitchell, Alexander R.; Coburn, Michael D.; Lee, Gregory S.; Schmidt, Robert D.; Pagoria, Philip F.; Hsu, Peter C.

    2006-06-06

    A method to convert surplus nitroarene explosives (picric acid, ammonium picrate,) into TATB is described. The process comprises three major steps: conversion of picric acid/ammonium picrate into picramide; conversion of picramide to TATB through vicarious nucleophilic substitution (VNS) of hydrogen chemistry; and purification of TATB.

  3. Purification and characterization of a hexanol-degrading enzyme extracted from apple

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An enzyme having activity towards n-hexanol was purified from apple and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 column. The obtained enzyme had a yi...

  4. Food additives

    MedlinePlus

    Food additives are substances that become part of a food product when they are added during the processing or making of that food. "Direct" food additives are often added during processing to: Add nutrients ...

  5. Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

    PubMed Central

    Pollock, Naomi; Cant, Natasha; Rimington, Tracy; Ford, Robert C.

    2014-01-01

    Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to <40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal. We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments. In this method, we provide comparison of the purification of CFTR in dodecyl-β-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity. PMID:24893839

  6. A simplified protocol for high-yield expression and purification of bacterial topoisomerase I.

    PubMed

    Jones, Jesse A; Price, Emily; Miller, Donovan; Hevener, Kirk E

    2016-08-01

    Type IA topoisomerases represent promising antibacterial drug targets. Data exists suggesting that the two bacterial type IA topoisomerase enzymes-topoisomerase I and topoisomerase III-share an overlapping biological role. Furthermore, topoisomerase I has been shown to be essential for the survival of certain organisms lacking topoisomerase III. With this in mind, it is plausible that topoisomerase I may represent a potential target for selective antibacterial drug development. As many reported bacterial topoisomerase I purification protocols have either suffered from relatively low yield, numerous steps, or a simple failure to report target protein yield altogether, a high-yield and high-purity bacterial topoisomerase I expression and purification protocol is highly desirable. The goal of this study was therefore to optimize the expression and purification of topoisomerase I from Streptococcus mutans, a clinically relevant organism that plays a significant role in oral and extra-oral infection, in order to quickly and easily attain the requisite quantities of pure target enzyme suitable for use in assay development, compound library screening, and carrying out further structural and biochemical characterization analyses. Herein we report the systematic implementation and analysis of various expression and purification techniques leading to the development and optimization of a rapid and straightforward protocol for the auto-induced expression and two-step, affinity tag purification of Streptococcus mutans topoisomerase I yielding >20 mg/L of enzyme at over 95% purity. PMID:27117979

  7. Food additives

    PubMed Central

    Spencer, Michael

    1974-01-01

    Food additives are discussed from the food technology point of view. The reasons for their use are summarized: (1) to protect food from chemical and microbiological attack; (2) to even out seasonal supplies; (3) to improve their eating quality; (4) to improve their nutritional value. The various types of food additives are considered, e.g. colours, flavours, emulsifiers, bread and flour additives, preservatives, and nutritional additives. The paper concludes with consideration of those circumstances in which the use of additives is (a) justified and (b) unjustified. PMID:4467857

  8. High Throughput Identification, Purification and Structural Characterization of Water Soluble Protein Complexes in Desulfovibrio vulgaris

    SciTech Connect

    Dong,, Ming; Han, Bong-Gyoon; Liu, Hui-Hai; Malik, J.; Geller, Jil; Yang, Li; Choi, M.; Chandonia, John-Marc; Arbelaez, Pablo; Sterling, H. J.; Typke, Dieter; Shatsky, Max; Brenner, Steve; Fisher, Susan; Williams, Evan; Szakal, Evelin; Allen, S.; Hall, S. C.; Hazen, Terry; Witkowska, H. E.; Jin, Jiming; Glaeser, Robert; Biggin, Mark

    2010-05-17

    Our scheme for the tagless purification of water soluble complexes. 10 g of protein from a crude bacterial extract is first fractionated by ammonium sulfate precipitation and then by a series of chromatographic steps: anion exchange (IEX), hydrophobic interaction (HIC), and finally size exclusion (Gel Filtration). Fractions from the last chromatography step are trypsin digested and peptides labeled with iTRAQ reagents to allow multiplexing and quantitation during mass spectrometric analysis. Elution profiles of identified proteins are then subjected to clustering analysis.

  9. Chromatography purification of canine adenoviral vectors.

    PubMed

    Segura, María Mercedes; Puig, Meritxell; Monfar, Mercè; Chillón, Miguel

    2012-06-01

    Canine adenovirus vectors (CAV2) are currently being evaluated for gene therapy, oncolytic virotherapy, and as vectors for recombinant vaccines. Despite the need for increasing volumes of purified CAV2 preparations for preclinical and clinical testing, their purification still relies on the use of conventional, scale-limited CsCl ultracentrifugation techniques. A complete downstream processing strategy for CAV2 vectors based on membrane filtration and chromatography is reported here. Microfiltration and ultra/diafiltration are selected for clarification and concentration of crude viral stocks containing both intracellular and extracellular CAV2 particles. A DNase digestion step is introduced between ultrafiltration and diafiltration operations. At these early stages, concentration of vector stocks with good recovery of viral particles (above 80%) and removal of a substantial amount of protein and nucleic acid contaminants is achieved. The ability of various chromatography techniques to isolate CAV2 particles was evaluated. Hydrophobic interaction chromatography using a Fractogel propyl tentacle resin was selected as a first chromatography step, because it allows removal of the bulk of contaminating proteins with high CAV2 yields (88%). An anion-exchange chromatography step using monolithic supports is further introduced to remove the remaining contaminants with good recovery of CAV2 particles (58-69%). The main CAV2 viral structural components are visualized in purified preparations by electrophoresis analyses. Purified vector stocks contained intact icosahedral viral particles, low contamination with empty viral capsids (10%), and an acceptable total-to-infectious particle ratio (below 30). The downstream processing strategy that was developed allows preparation of large volumes of high-quality CAV2 stocks. PMID:22799886

  10. Expression in E. coli and purification of Thermus thermophilus translation initiation factors IF1 and IF3.

    PubMed

    Wolfrum, Alexandra; Brock, Stephan; Mac, Thi; Grillenbeck, Norbert

    2003-05-01

    The initiation of protein translation in bacteria requires in addition to mRNA, fMet-tRNA, and ribosomal subunits three protein factors, the initiation factor 1 (IF1), initiation factor 2 (IF2), and initiation factor 3 (IF3). The genes coding for IF1 and IF3 from Thermus thermophilus have been identified and cloned into pET expression vector and were expressed as soluble proteins in Escherichia coli. IF1 was purified by a DEAE-cellulose chromatography, followed by heat denaturation, chromatography on Hydroxylapatit, and gel permeation chromatography using Sephacryl 200HR. For the purification of IF3, a heat denaturation step is followed by anion-exchange chromatography on Q-Sepharose FF and gel permeation chromatography on Sephacryl 200HR. Using these procedures we obtained chromatographically pure and biologically active preparations of both T. thermophilus IF1 and IF3. PMID:12729721

  11. Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use

    PubMed Central

    Šiman, Pavel; Filipová, Alžběta; Tichá, Alena; Niang, Mohamed; Bezrouk, Aleš; Havelek, Radim

    2016-01-01

    A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use. PMID:27152419

  12. Efficient expression, purification, and characterization of a novel FAD-dependent glucose dehydrogenase from Aspergillus terreus in Pichia pastoris.

    PubMed

    Yang, Yufeng; Huang, Lei; Wang, Jufang; Wang, Xiaoning; Xu, Zhinan

    2014-11-28

    Flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) can utilize a variety of external electron acceptors and also has stricter substrate specificity than any other glucose oxidoreductases, which makes it the ideal diagnostic enzyme in the field of glucose biosensors. A gene coding for a hypothetical protein, similar to glucose oxidase and derived from Aspergillus terreus NIH2624, was overexpressed in Pichia pastoris GS115 under the control of an AOX1 promoter with a level of 260,000 U/l in the culture supernatant after fed-batch cultivation for 84 h. After a three-step purification protocol that included isopropanol precipitation, affinity chromatography, and a second isopropanol precipitation, recombinant FAD-GDH was purified with a recovery of 65%. This is the first time that isopropanol precipitation has been used to concentrate a fermentation supernatant and exchange buffers after affinity chromatography purification. The purified FAD-GDH exhibited a broad and diffuse band between 83 and 150 kDa. The recombinant FAD-GDH was stable across a wide pH range (3.5 to 9.0) with maximum activity at pH 7.5 and 55°C. In addition, it displayed very high thermal stability, with a half-life of 82 min at 60°C. These characteristics indicate that FAD-GDH will be useful in the field of glucose biosensors. PMID:25022525

  13. Dodecyl Maltopyranoside Enabled Purification of Active Human GABA Type A Receptors for Deep and Direct Proteomic Sequencing*

    PubMed Central

    Zhang, Xi; Miller, Keith W.

    2015-01-01

    The challenge in high-quality membrane proteomics is all about sample preparation prior to HPLC, and the cell-to-protein step poses a long-standing bottleneck. Traditional protein extraction methods apply ionic or poly-disperse detergents, harsh denaturation, and repeated protein/peptide precipitation/resolubilization afterward, but suffer low yield, low reproducibility, and low sequence coverage. Contrary to attempts to subdue, we resolved this challenge by providing proteins nature-and-activity-promoting conditions throughout preparation. Using 285-kDa hetero-pentameric human GABA type A receptor overexpressed in HEK293 as a model, we describe a n-dodecyl-β-d-maltopyranoside/cholesteryl hemisuccinate (DDM/CHS)-based affinity purification method, that produced active receptors, supported protease activity, and allowed high performance with both in-gel and direct gel-free proteomic analyses—without detergent removal. Unlike conventional belief that detergents must be removed before HPLC MS, the high-purity low-dose nonionic detergent DDM did not interfere with peptides, and obviated removal or desalting. Sonication or dropwise addition of detergent robustly solubilized over 90% of membrane pellets. The purification conditions were comparable to those applied in successful crystallizations of most membrane proteins. These results enabled streamlined proteomics of human synaptic membrane proteins, and more importantly, allowed directly coupling proteomics with crystallography to characterize both static and dynamic structures of membrane proteins in crystallization pipelines. PMID:25473089

  14. Effective Method of Purification of Betulin from Birch Bark: The Importance of Its Purity for Scientific and Medicinal Use.

    PubMed

    Šiman, Pavel; Filipová, Alžběta; Tichá, Alena; Niang, Mohamed; Bezrouk, Aleš; Havelek, Radim

    2016-01-01

    A new and relatively simple method for purification of betulin from birch bark extract was developed in this study. Its five purification steps are based on the differential solubility of extract components in various solvents and their crystallization and/or precipitation, on their affinity for Ca(OH)2 in ethanol, and on the affinity of some impurities for silica gel in chloroform. In addition, all used solvents can be simply recycled. Betulin of more than 99% purity can be prepared by this method with minimal costs. Various observations including crystallization of betulin, changes in crystals during heating, and attempt of localization of betulin in outer birch bark are also described in this work. The original extract, fraction without betulinic acid and lupeol, amorphous fraction of pure betulin, final crystalline fraction of pure betulin and commercial betulin as a standard were employed to determine the antiproliferative/cytotoxic effect. We used WST-1 tetrazolium-based assays with triple negative breast cancer cell line BT-549. The decrease in cell survival showed clear relationship with the purity of the samples, being most pronounced using our final product of pure crystalline betulin. WST-1 proliferation/cytotoxicity test using triple negative breast cancer cell line BT-549 clearly showed the importance of purity of betulin for biological experiments and, apparently, for its medicinal use. PMID:27152419

  15. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  16. Purification of a Multidrug Resistance Transporter for Crystallization Studies

    PubMed Central

    Alegre, Kamela O.; Law, Christopher J.

    2015-01-01

    Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

  17. Purification and concentration of DNA from aqueous solutions.

    PubMed

    Moore, D

    2001-05-01

    This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated for reasons of convenience. The Basic Protocol, using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for the purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols provide modifications to the basic protocol that are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low-molecular-weight oligonucleotides and triphosphates. PMID:18428438

  18. Purification of a Multidrug Resistance Transporter for Crystallization Studies.

    PubMed

    Alegre, Kamela O; Law, Christopher J

    2015-01-01

    Crystallization of integral membrane proteins is a challenging field and much effort has been invested in optimizing the overexpression and purification steps needed to obtain milligram amounts of pure, stable, monodisperse protein sample for crystallography studies. Our current work involves the structural and functional characterization of the Escherichia coli multidrug resistance transporter MdtM, a member of the major facilitator superfamily (MFS). Here we present a protocol for isolation of MdtM to increase yields of recombinant protein to the milligram quantities necessary for pursuit of structural studies using X-ray crystallography. Purification of MdtM was enhanced by introduction of an elongated His-tag, followed by identification and subsequent removal of chaperonin contamination. For crystallization trials of MdtM, detergent screening using size exclusion chromatography determined that decylmaltoside (DM) was the shortest-chain detergent that maintained the protein in a stable, monodispersed state. Crystallization trials of MdtM performed using the hanging-drop diffusion method with commercially available crystallization screens yielded 3D protein crystals under several different conditions. We contend that the purification protocol described here may be employed for production of high-quality protein of other multidrug efflux members of the MFS, a ubiquitous, physiologically and clinically important class of membrane transporters. PMID:27025617

  19. Purification of large plasmids with methacrylate monolithic columns.

    PubMed

    Krajnc, Nika Lendero; Smrekar, Franci; Cerne, Jasmina; Raspor, Peter; Modic, Martina; Krgovic, Danijela; Strancar, Ales; Podgornik, Ales

    2009-08-01

    The rapid evolution of gene therapy and DNA vaccines results in an increasing interest in producing large quantities of pharmaceutical grade plasmid DNA. Most current clinical trials involve plasmids of 10 kb or smaller in size, however, future requirements for multigene vectors including extensive control regions may require the production of larger plasmids, e. g., 20 kb and bigger. The objective of this study was to examine certain process conditions for purification of large plasmids with the size of up to 93 kb. Since there is a lack of knowledge about production and purification of bigger plasmid DNA, cell lysis and storage conditions were investigated. The impact of chromatographic system and methacrylate monolithic column on the degradation of plasmid molecules under nonbinding conditions at different flow rates was studied. Furthermore, capacity measurements varying salt concentration in loading buffer were performed and the capacities up to 13 mg of plasmid per mL of the monolithic column were obtained. The capacity flow independence in the range from 130 to 370 cm/h was observed. Using high resolution monolithic column the separation of linear and supercoiled isoforms of large plasmids was obtained. Last but not least, since the baseline separation of RNA and pDNA was achieved, the one step purification on larger CIM DEAE 8 mL tube monolithic column was performed and the fractions were analyzed by CIM analytical monolithic columns. PMID:19598166

  20. Development of EV71 virus-like particle purification processes.

    PubMed

    Lin, Shih-Yeh; Chiu, Hsin-Yi; Chiang, Bor-Luen; Hu, Yu-Chen

    2015-11-01

    Enterovirus 71 (EV71) causes the outbreaks of hand-foot-and-mouth disease and results in deaths of hundreds of young children. EV71 virus-like particles (VLPs) are empty capsids consisting of viral structural proteins and can elicit potent immune responses, thus holding promise as an EV71 vaccine candidate. However, an efficient, scalable production and purification scheme is missing. For mass production of EV71 VLPs, this study aimed to develop a production and chromatography-based purification process. We first demonstrated the successful EV71 VLPs production in the stirred-tank bioreactor in which High Five™ cells were infected with a recombinant baculovirus co-expressing EV71 structural polyprotein P1 and protease 3CD. The culture supernatant containing the VLPs was subjected to tangential flow filtration (TFF) for concentration/diafiltration, which enabled the removal of >80% of proteins while recovering >80% of VLPs. The concentrated VLPs were next subjected to hydroxyapatite chromatography (HAC) in which the VLPs were mainly found in the flow through. After another TFF concentration/diafiltration, the VLPs were purified by size-exclusion chromatography (SEC) and concentrated/diafiltered by a final TFF. The integrated process yielded an overall VLPs recovery of ≈ 36% and a purity of ≈ 83%, which was better or comparable to the recovery and purity for the purification of live EV71 virus particles. This process thus may move the EV71 VLPs vaccine one step closer to the clinical applications. PMID:25939279

  1. Simple method for purification of enterotoxigenic Escherichia coli fimbriae.

    PubMed

    Curtis, Brittany; Grassel, Christen; Laufer, Rachel S; Sears, Khandra T; Pasetti, Marcela F; Barry, Eileen M; Simon, Raphael

    2016-03-01

    Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera. PMID:26581778

  2. Purification of phosphatidylinositol kinase from bovine brain myelin.

    PubMed Central

    Saltiel, A R; Fox, J A; Sherline, P; Sahyoun, N; Cuatrecasas, P

    1987-01-01

    A membrane-bound phosphatidylinositol (PI) kinase (EC 2.7.1.67) was purified by affinity chromatography from bovine brain myelin. This enzyme activity was solubilized with non-ionic detergent and chromatographed on an anion-exchange column. Further purification was achieved by affinity chromatography on PI covalently coupled to epoxy-activated Sepharose, which was eluted with a combination of PI and detergent. The final step in the purification was by gel filtration on an Ultrogel AcA44 column. This procedure afforded greater than 5500-fold purification of the enzyme from whole brain myelin. The resulting activity exhibited a major silver-stained band on SDS/polyacrylamide-gel electrophoresis with an apparent Mr 45,000. The identity of this band as PI kinase was corroborated by demonstration of enzyme activity in the gel region corresponding to that of the stained protein. The purified enzyme exhibited a non-linear dependence on PI as substrate, with two apparent kinetic components. The lower-affinity component exhibited a Km similar to that observed for the phosphorylation of phosphatidylinositol 4-phosphate by the enzyme. PMID:3036072

  3. Large-Scale Functional Purification of Recombinant HIV-1 Capsid

    PubMed Central

    Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M.; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  4. Large-scale functional purification of recombinant HIV-1 capsid.

    PubMed

    Hung, Magdeleine; Niedziela-Majka, Anita; Jin, Debi; Wong, Melanie; Leavitt, Stephanie; Brendza, Katherine M; Liu, Xiaohong; Sakowicz, Roman

    2013-01-01

    During human immunodeficiency virus type-1 (HIV-1) virion maturation, capsid proteins undergo a major rearrangement to form a conical core that protects the viral nucleoprotein complexes. Mutations in the capsid sequence that alter the stability of the capsid core are deleterious to viral infectivity and replication. Recently, capsid assembly has become an attractive target for the development of a new generation of anti-retroviral agents. Drug screening efforts and subsequent structural and mechanistic studies require gram quantities of active, homogeneous and pure protein. Conventional means of laboratory purification of Escherichia coli expressed recombinant capsid protein rely on column chromatography steps that are not amenable to large-scale production. Here we present a function-based purification of wild-type and quadruple mutant capsid proteins, which relies on the inherent propensity of capsid protein to polymerize and depolymerize. This method does not require the packing of sizable chromatography columns and can generate double-digit gram quantities of functionally and biochemically well-behaved proteins with greater than 98% purity. We have used the purified capsid protein to characterize two known assembly inhibitors in our in-house developed polymerization assay and to measure their binding affinities. Our capsid purification procedure provides a robust method for purifying large quantities of a key protein in the HIV-1 life cycle, facilitating identification of the next generation anti-HIV agents. PMID:23472130

  5. Mimiviruses: Replication, Purification, and Quantification.

    PubMed

    Abrahão, Jônatas Santos; Oliveira, Graziele Pereira; Ferreira da Silva, Lorena Christine; Dos Santos Silva, Ludmila Karen; Kroon, Erna Geessien; La Scola, Bernard

    2016-01-01

    The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc. PMID:27153385

  6. One-step cell lysis suitable for quantitative bacteria detection in inhibitor-laden sands

    NASA Astrophysics Data System (ADS)

    Lim, Hyun Jeong; Choi, Jung-Hyun; Son, Ahjeong

    2015-04-01

    Complexity and heterogeneity of soils often hinder effective DNA extraction from the soil matrix. In particular, conventional DNA extraction techniques require extensive purification which makes DNA extraction time-consuming and labor-intensive. Other drawbacks include lower recovery yield, degradation, and damage of DNA, which are also caused by intensive purifications during DNA extraction. Therefore a rapid and simple and yet effective DNA pretreatment method is preferred for environmental monitoring and screening. This study has evaluated the feasibility of simple physical pretreatment for effective cell lysis of bacteria in sands. Bead beating method was selected as an effective physical cell lysis method in this study. We examined the capability of this physical lysis for Pseudomonas putida seeded sands without additional chemical purification steps. The lysate from the method was analysed by the quantitative polymerase chain reaction (qPCR) assay and subsequently compared to that by commercial DNA extraction kit. The best lysis condition (treatment with 0.1 mm glass beads at 3000 rpm for 3 minutes) was selected. The qPCR results of bead beating treated samples showed the better performance than that of conventional DNA extraction kit. Moreover, the qPCR assay was performed to the sands laden with qPCR inhibitors (humic acids, clay, and magnesium), which generally present in environmental samples. Further experiments with the sands containing less than 10 μg/g of humic acids and 70% of clay showed successful quantification results of qPCR assay. In conclusion, the bead beating method is useful for simplified DNA extraction prior to qPCR analysis for sand samples of particular composition. It is expected that this approach will be beneficial for environmental in-situ analysis or immediate pre-screening. It also provides the groundwork for future studies with real soil samples that have various physico-chemical properties.

  7. Chromatographic purification and size separation of carbon nanotubes

    NASA Astrophysics Data System (ADS)

    Duesberg, G. S.; Muster, J.; Krstic, V.; Burghard, M.; Roth, S.

    1998-08-01

    The efficient purification of single-wall and multi-wall carbon nanotubes (NTs) by columnar size exclusion chromatography (SEC) is reported. In this process, carbon nanospheres (polyhedra), amorphous carbon and metal particles are removed from aqueous surfactant-stabilised dispersions of NT raw material. TEM and AFM investigations revealed that more than 40-50% of the purified material consists of individual tubes. In addition, length separation of the tubes is achieved.

  8. Water purification using organic salts

    DOEpatents

    Currier, Robert P.

    2004-11-23

    Water purification using organic salts. Feed water is mixed with at least one organic salt at a temperature sufficiently low to form organic salt hydrate crystals and brine. The crystals are separated from the brine, rinsed, and melted to form an aqueous solution of organic salt. Some of the water is removed from the aqueous organic salt solution. The purified water is collected, and the remaining more concentrated aqueous organic salt solution is reused.

  9. Melting And Purification Of Niobium

    SciTech Connect

    Salles Moura, Hernane R.; Moura, Lourenco de

    2007-08-09

    The aspects involved in the purification of niobium in Electron Beam Furnaces will be outlined and correlated with practical experience accumulated over 17 years of continuously producing high purity niobium metal and niobium-zirconium ingots at CBMM, meeting the needs for a wide range of uses. This paper also reports some comments regarding raw material requirements, the experience on cold hearth operation melting niobium and the production of large grains niobium ingots by CBMM with some comments of their main characteristics.

  10. Melting And Purification Of Niobium

    NASA Astrophysics Data System (ADS)

    Moura, Hernane R. Salles; de Moura, Lourenço

    2007-08-01

    The aspects involved in the purification of niobium in Electron Beam Furnaces will be outlined and correlated with practical experience accumulated over 17 years of continuously producing high purity niobium metal and niobium-zirconium ingots at CBMM, meeting the needs for a wide range of uses. This paper also reports some comments regarding raw material requirements, the experience on cold hearth operation melting niobium and the production of large grains niobium ingots by CBMM with some comments of their main characteristics.

  11. Radon assay and purification techniques

    SciTech Connect

    Simgen, Hardy

    2013-08-08

    Radon is a source of background in many astroparticle physics experiments searching for rare low energy events. In this paper an overview about radon in the field is given including radon detection techniques, radon sources and material screening with respect to radon emanation. Finally, also the problem of long-lived radioactive {sup 222}Rn-daughters and the question of gas purification from radon is addressed.

  12. Robotic high-throughput purification of affinity-tagged recombinant proteins.

    PubMed

    Wiesler, Simone C; Weinzierl, Robert O J

    2015-01-01

    Affinity purification of recombinant proteins has become the method of choice to obtain good quantities and qualities of proteins for a variety of downstream biochemical applications. While manual or FPLC-assisted purification techniques are generally time-consuming and labor-intensive, the advent of high-throughput technologies and liquid handling robotics has simplified and accelerated this process significantly. Additionally, without the human factor as a potential source of error, automated purification protocols allow for the generation of large numbers of proteins simultaneously and under directly comparable conditions. The delivered material is ideal for activity comparisons of different variants of the same protein. Here, we present our strategy for the simultaneous purification of up to 24 affinity-tagged proteins for activity measurements in biochemical assays. The protocol described is suitable for the scale typically required in individual research laboratories. PMID:25749949

  13. SNO+ Scintillator Purification and Assay

    SciTech Connect

    Ford, R.; Vazquez-Jauregui, E.; Chen, M.; Chkvorets, O.; Hallman, D.

    2011-04-27

    We describe the R and D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O{sub 2}, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed ''natural'' radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  14. SNO+ Scintillator Purification and Assay

    NASA Astrophysics Data System (ADS)

    Ford, R.; Chen, M.; Chkvorets, O.; Hallman, D.; Vázquez-Jáuregui, E.

    2011-04-01

    We describe the R&D on the scintillator purification and assay methods and technology for the SNO+ neutrino and double-beta decay experiment. The SNO+ experiment is a replacement of the SNO heavy water with liquid scintillator comprised of 2 g/L PPO in linear alkylbenzene (LAB). During filling the LAB will be transported underground by rail car and purified by multi-stage distillation and steam stripping at a flow rate of 19 LPM. While the detector is operational the scintillator can be recirculated at 150 LPM (full detector volume in 4 days) to provide repurification as necessary by either water extraction (for Ra, K, Bi) or by functional metal scavenger columns (for Pb, Ra, Bi, Ac, Th) followed by steam stripping to remove noble gases and oxygen (Rn, O2, Kr, Ar). The metal scavenger columns also provide a method for scintillator assay for ex-situ measurement of the U and Th chain radioactivity. We have developed "natural" radioactive spikes of Pb and Ra in LAB and use these for purification testing. Lastly, we present the planned operating modes and purification strategies and the plant specifications and design.

  15. Technological assumptions for biogas purification.

    PubMed

    Makareviciene, Violeta; Sendzikiene, Egle

    2015-01-01

    Biogas can be used in the engines of transport vehicles and blended into natural gas networks, but it also requires the removal of carbon dioxide, hydrogen sulphide, and moisture. Biogas purification process flow diagrams have been developed for a process enabling the use of a dolomite suspension, as well as for solutions obtained by the filtration of the suspension, to obtain biogas free of hydrogen sulphide and with a carbon dioxide content that does not exceed 2%. The cost of biogas purification was evaluated on the basis of data on biogas production capacity and biogas production cost obtained from local water treatment facilities. It has been found that, with the use of dolomite suspension, the cost of biogas purification is approximately six times lower than that in the case of using a chemical sorbent such as monoethanolamine. The results showed travelling costs using biogas purified by dolomite suspension are nearly 1.5 time lower than travelling costs using gasoline and slightly lower than travelling costs using mineral diesel fuel. PMID:25609385

  16. Rapid separation and purification of uranium and plutonium from dilute-matrix samples

    DOE PAGESBeta

    Armstrong, Christopher R.; Ticknor, Brian W.; Hall, Gregory; Cadieux, James R.

    2014-03-11

    This work presents a streamlined separation and purification approach for trace uranium and plutonium from dilute (carrier-free) matrices. The method, effective for nanogram quantities of U and femtogram to picogram quantities of Pu, is ideally suited for environmental swipe samples that contain a small amount of collected bulk material. As such, it may be applicable for processing swipe samples such as those collected in IAEA inspection activities as well as swipes that are loaded with unknown analytes, such as those implemented in interlaboratory round-robin or proficiency tests. Additionally, the simplified actinide separation could find use in internal laboratory monitoring ofmore » clean room conditions prior to or following more extensive chemical processing. We describe key modifications to conventional techniques that result in a relatively rapid, cost-effective, and efficient U and Pu separation process. We demonstrate the efficacy of implementing anion exchange chromatography in a single column approach. We also show that hydrobromic acid is an effective substitute in lieu of hydroiodoic acid for eluting Pu. Lastly, we show that nitric acid is an effective digestion agent in lieu of perchloric acid and/or hydrofluoric acid. A step by step procedure of this process is detailed.« less

  17. Rapid separation and purification of uranium and plutonium from dilute-matrix samples

    SciTech Connect

    Armstrong, Christopher R.; Ticknor, Brian W.; Hall, Gregory; Cadieux, James R.

    2014-03-11

    This work presents a streamlined separation and purification approach for trace uranium and plutonium from dilute (carrier-free) matrices. The method, effective for nanogram quantities of U and femtogram to picogram quantities of Pu, is ideally suited for environmental swipe samples that contain a small amount of collected bulk material. As such, it may be applicable for processing swipe samples such as those collected in IAEA inspection activities as well as swipes that are loaded with unknown analytes, such as those implemented in interlaboratory round-robin or proficiency tests. Additionally, the simplified actinide separation could find use in internal laboratory monitoring of clean room conditions prior to or following more extensive chemical processing. We describe key modifications to conventional techniques that result in a relatively rapid, cost-effective, and efficient U and Pu separation process. We demonstrate the efficacy of implementing anion exchange chromatography in a single column approach. We also show that hydrobromic acid is an effective substitute in lieu of hydroiodoic acid for eluting Pu. Lastly, we show that nitric acid is an effective digestion agent in lieu of perchloric acid and/or hydrofluoric acid. A step by step procedure of this process is detailed.

  18. Use of Tandem Affinity Chromatography for Purification of Cannabinoid Receptor CB2

    PubMed Central

    Locatelli-Hoops, Silvia C.; Yeliseev, Alexei A.

    2016-01-01

    Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor. PMID:24943318

  19. A membrane-based purification process for cell culture-derived influenza A virus.

    PubMed

    Weigel, Thomas; Solomaier, Thomas; Wehmeyer, Sebastian; Peuker, Alessa; Wolff, Michael W; Reichl, Udo

    2016-02-20

    A simple membrane-based purification process for cell culture-derived influenza virus was established that relies on only two chromatographic unit operations to achieve the contamination limits required according to regulatory authorities. After clarification and concentration, a pseudo-affinity membrane adsorber (sulfated cellulose, SCMA) was applied for virus capture. The subsequent polishing step consisted of a salt-tolerant anion exchange membrane adsorber (STMA) to bind residual DNA. For the presented process neither a buffer exchange step nor a nuclease step for further DNA digestion were required. As a starting point, a two-salt strategy (including a polyvalent ion) was employed to screen STMA conditions in a 96-well plate format. After optimization on chromatographic laboratory scale, the virus recovery was up to 97% with a residual DNA level below 0.82%. In addition, the STMA was characterized regarding its dynamic binding capacity and the impact of flow rate on yields and contamination levels. Overall, the total virus yield for influenza virus A/PR/8/34 (H1/N1) of this two-step membrane process was 75%, while the protein and the DNA contamination level could be reduced to 24% and at least 0.5%, respectively. With 19.8μg protein and 1.2ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived vaccines for human use. Overall, the presented downstream process might serve as a generic and economic platform technology for production of cell culture-derived viruses and viral vectors. PMID:26712479

  20. Isolation of 1E4 IgM Anti-Fasciola hepatica Rediae Monoclonal Antibody from Ascites: Comparison of Two Purification Protocols.

    PubMed

    Alba, Annia; Marcet, Ricardo; Otero, Oscar; Hernández, Hilda M; Figueredo, Mabel; Sarracent, Jorge

    2016-02-01

    Purification of immunoglobulin M (IgM) antibodies could be challenging, and is often characterized by the optimization of the purification protocol to best suit the particular features of the molecule. Here, two different schemes were compared to purify, from ascites, the 1E4 IgM monoclonal antibody (mAb) previously raised against the stage of redia of the trematode Fasciola hepatica. This immunoglobulin is used as capture antibody in an immunoenzymatic assay to detect parasite ongoing infection in its intermediate hosts. The first purification protocol of the 1E4 mAb involved two chromatographic steps: an affinity chromatography on a Concanavalin A matrix followed by size exclusion chromatography. An immunoaffinity chromatography was selected as the second protocol for one-step purification of the antibody using the crude extract of adult parasites coupled to a commercial matrix. Immunoreactivity of the fractions during purification schemes was assessed by indirect immunoenzymatic assays against the crude extract of F. hepatica rediae, while purity was estimated by protein electrophoresis. Losses on the recovery of the antibody isolated by the first purification protocol occurred due to protein precipitation during the concentration of the sample and to low resolution of the size exclusion molecular chromatography step regarding this particular immunoglobulin. The immunoaffinity chromatography using F. hepatica antigens as ligands proved to be the most suitable protocol yielding a pure and immunoreactive antibody. The purification protocols used are discussed regarding efficiency and difficulties. PMID:26828226

  1. Stepping motor controller

    DOEpatents

    Bourret, S.C.; Swansen, J.E.

    1982-07-02

    A stepping motor is microprocessor controlled by digital circuitry which monitors the output of a shaft encoder adjustably secured to the stepping motor and generates a subsequent stepping pulse only after the preceding step has occurred and a fixed delay has expired. The fixed delay is variable on a real-time basis to provide for smooth and controlled deceleration.

  2. Stepping motor controller

    DOEpatents

    Bourret, Steven C.; Swansen, James E.

    1984-01-01

    A stepping motor is microprocessingly controlled by digital circuitry which monitors the output of a shaft encoder adjustably secured to the stepping motor and generates a subsequent stepping pulse only after the preceding step has occurred and a fixed delay has expired. The fixed delay is variable on a real-time basis to provide for smooth and controlled deceleration.

  3. Food additives.

    PubMed

    Berglund, F

    1978-01-01

    The use of additives to food fulfils many purposes, as shown by the index issued by the Codex Committee on Food Additives: Acids, bases and salts; Preservatives, Antioxidants and antioxidant synergists; Anticaking agents; Colours; Emulfifiers; Thickening agents; Flour-treatment agents; Extraction solvents; Carrier solvents; Flavours (synthetic); Flavour enhancers; Non-nutritive sweeteners; Processing aids; Enzyme preparations. Many additives occur naturally in foods, but this does not exclude toxicity at higher levels. Some food additives are nutrients, or even essential nutritents, e.g. NaCl. Examples are known of food additives causing toxicity in man even when used according to regulations, e.g. cobalt in beer. In other instances, poisoning has been due to carry-over, e.g. by nitrate in cheese whey - when used for artificial feed for infants. Poisonings also occur as the result of the permitted substance being added at too high levels, by accident or carelessness, e.g. nitrite in fish. Finally, there are examples of hypersensitivity to food additives, e.g. to tartrazine and other food colours. The toxicological evaluation, based on animal feeding studies, may be complicated by impurities, e.g. orthotoluene-sulfonamide in saccharin; by transformation or disappearance of the additive in food processing in storage, e.g. bisulfite in raisins; by reaction products with food constituents, e.g. formation of ethylurethane from diethyl pyrocarbonate; by metabolic transformation products, e.g. formation in the gut of cyclohexylamine from cyclamate. Metabolic end products may differ in experimental animals and in man: guanylic acid and inosinic acid are metabolized to allantoin in the rat but to uric acid in man. The magnitude of the safety margin in man of the Acceptable Daily Intake (ADI) is not identical to the "safety factor" used when calculating the ADI. The symptoms of Chinese Restaurant Syndrome, although not hazardous, furthermore illustrate that the whole ADI

  4. An Additional Step in the Guided Lecture Procedure.

    ERIC Educational Resources Information Center

    Toole, Robert J.

    2000-01-01

    Describes the Guided Lecture Procedure (GLP), a procedure that requires students to suspend all notetaking and listen carefully during an approximately 20-minute lecture, followed by an active notetaking and small group interaction phase. Adds one extra requirement in the active notetaking phase: requiring each learner to write a question for the…

  5. Large-scale monoclonal antibody purification by continuous chromatography, from process design to scale-up.

    PubMed

    Girard, Valérie; Hilbold, Nicolas-Julian; Ng, Candy K S; Pegon, Laurence; Chahim, Wael; Rousset, Fabien; Monchois, Vincent

    2015-11-10

    The development and optimization of a purification process of monoclonal antibodies based on two continuous chromatography steps for capture and intermediate purification are presented. The two chromatography steps were individually optimized using either batch chromatography or sequential multicolumn chromatography (SMCC). Proprietary simulation software was used to optimize SMCC and to evaluate the potential gains compared with batch chromatography. The SMCC recipes provided by the simulation software were evaluated experimentally. A good correlation was found between the simulated results and experimental observations. Significant gains were observed on the productivity, buffer consumption and the volume of resin required for SMCC over batch chromatography. Based on these results, a chained process from the capture to polishing steps was implemented. This chained process demonstrated significantly better performance compared with the batch equivalent while satisfying the specifications. The expected positive impact provided by implementing continuous chromatography is also discussed. PMID:25962790

  6. Production of horsegram (Dolichos biflorus) Bowman-Birk inhibitor by an intein mediated protein purification system.

    PubMed

    Kumar, Vinod; Gowda, Lalitha R

    2013-05-01

    The seeds of the legume horsegram (Dolichos biflorus), a protein rich pulse (bean), contain multiple forms of Bowman-Birk inhibitors (protease inhibitors). The major inhibitor HGI-III contains seven interweaving disulfides and is extremely stable to high temperatures. A soluble HGI-III (rHGI) with the native N-terminus was produced using a pTWIN IMPACT™ purification system. Yield of rHGI was improved by introducing a trypsin sepharose affinity chromatography step resulting in ∼670 fold purification. The biochemical characteristics of rHGI point to its close similarity to seed HGI-III not only in its structure but also in its inhibitory characteristics toward bovine trypsin and chymotrypsin. The expression and purification strategy presented here promises to produce BBIs in their natural form for pharmacological and therapeutic use. PMID:23422783

  7. Precipitation of filamentous bacteriophages for their selective recovery in primary purification.

    PubMed

    Branston, Steven; Stanley, Emma; Keshavarz-Moore, Eli; Ward, John

    2012-01-01

    Filamentous bacteriophages and their derivatives are showing great promise as a whole new class of industrial agents, such as biologically based nano-materials and viral vectors. This raises challenges for their large-scale manufacture, principally due to the lack of bioprocessing knowledge. This article addresses what will be a potentially important option in the primary purification of the bacteriophages. Polyethylene glycol (PEG)-salt dual precipitants, calcium ions, spermidine, and isoelectric precipitation were first examined for their potential suitability for bacteriophage concentration under both pure and broth conditions. Successful precipitants were further studied on the basis of their selective purification ability from DNA and protein contaminants in a clarified broth system. Both PEG-based and isoelectric precipitations resulted in bacteriophage purity improvements, and PEG-based precipitations offered the highest selectivities. This work shows that precipitation of bacteriophages can be an effective primary purification step in a large-scale bioprocess. PMID:21905275

  8. Development of a novel affinity chromatography resin for platform purification of lambda fabs.

    PubMed

    Eifler, Nora; Medaglia, Giovanni; Anderka, Oliver; Laurin, Linus; Hermans, Pim

    2014-01-01

    Antigen-binding fragments (Fabs) are novel formats in the growing pipeline of biotherapeutics. Sharing similar features to monoclonal antibodies (mAbs) with regard to expression, Fabs are considered as unchallenging for upstream development. Yet for downstream processing, the mature mAb downstream purification platform is not directly applicable. New approaches need to be found to achieve a lean purification process that maintains quality, productivity, and timelines while being generically applicable independent of the expression system. In a successful collaboration, BAC BV, GE Healthcare, and Novartis Pharma AG have developed a new affinity chromatography medium (resin) suitable to support cGMP manufacturing of lambda Fabs. We show that using this novel chromatography medium for the capture step, a purification platform for lambda Fabs can be established. PMID:25082738

  9. Purification of Tronoh Silica Sand via preliminary process of mechanical milling

    NASA Astrophysics Data System (ADS)

    H, Nazratulhuda; M, Othman

    2016-02-01

    The purification of Tronoh silica sand is an important step in expanding technical applications of this silica sand. However no research on purifying of Tronoh silica sand has been reported. This study is focused on ball milling technique as a preliminary technique for Tronoh silica sand purification. The objectives are to study the effect of ball milling to the purification of the silica sand and to analyze its characteristics after the ball milling process. The samples before and after milling process were analyzed by using XRF, XRD, SEM and TEM. Results showed that the purity of SiO2 was increased, the size of the particles has been reduced and the surface area has increased. The crystalline phases for the silica before and after 4 hour milling time were remained constant.

  10. Overlimiting current and water purification in porous materials

    NASA Astrophysics Data System (ADS)

    Deng, Daosheng; Aouad, Wassim; Schlumpberger, Sven; Bazant, Martin Z.

    2012-11-01

    Salt transport in bulk electrolytes occurs by diffusion and convection, but in microfluidic devices and porous media, the presence of charged side walls leads to additional surface transport mechanisms, surface conduction and electro-osmotic flows, which become more important as the bulk salt concentration decreases. As a result, it is possible to exceed the diffusion-limited current to a membrane or electrode. In this work, we present experimental observations of over-limiting current to an ion-exchange membrane through a porous glass frit with submicron pores. Under this operation conditions, we also demonstrate the continuous extraction of depleted solution for water purification, including removing heavy metal ions, filtrating aggregated particles and reducing dye concentration. The porous media pave the way for practical water desalination and purification.

  11. Liquid xenon purification, de-radonation (and de-kryptonation)

    SciTech Connect

    Pocar, Andrea

    2015-08-17

    Liquid xenon detectors are at the forefront of rare event physics, including searches for neutrino-less double beta decay and WIMP dark matter. The xenon for these experiments needs to be purified from chemical impurities such as electronegative atoms and molecules, which absorb ionization electrons, and VUV (178 nm) scintillation light-absorbing chemical species. In addition, superb purification from radioactive impurities is required. Particularly challenging are radioactive noble isotopes ({sup 85}Kr,{sup 39,42}Ar,{sup 220,222}Rn). Radon is a particularly universal problem, due to the extended decay sequence of its daughters and its ubiquitous presence in detector materials. Purification and de-radonation of liquid xenon are addressed with particular focus on the experience gained with the EXO-200 neutrino-less double beta decay detector.

  12. Development of an Immunoaffinity Method for Purification of Streptokinase

    PubMed Central

    Karimi, Zohreh; Babashamsi, Mohammad; Asgarani, Ezat; Salimi, Ali

    2012-01-01

    Background Streptokinase is a potent activator of plasminogen to plasmin, the enzyme that can solubilize the fibrin network in blood clots. Streptokinase is currently used in clinical medicine as a thrombolytic agent. It is naturally secreted by β-hemolytic streptococci. Methods To reach an efficient method of purification, an immunoaffinity chromatography method was developed that could purify the streptokinase in a single step with high yield. At the first stage, a CNBr-Activated sepharose 4B-Lysine column was made to purify the human blood plasminogen. The purified plasminogen was utilized to construct a column that could purify the streptokinase. The rabbit was immunized with the purified streptokinase and the anti-streptokinase (IgG) purified on another streptokinase substituted sepharose-4B column. The immunoaffinity column was developed by coupling the purified anti-Streptokinase (IgG) to sepharose 6MB–Protein A. The Escherichia coli (E.coli) BL21 (DE3) pLysS strain was transformed by the recombinant construct (cloned streptokinase gene in pGEX-4T-2 vector) and gene expression was induced by IPTG. The expressed protein was purified by immunoaffinity chromatography in a single step. Results The immunoaffinity column could purify the recombinant fusion GST-SK to homogeneity. The purity of streptokinase was confirmed by SDS-PAGE as a single band of about 71 kD and its biological activity determined in a specific streptokinase assay. The yield of the purification was about 94%. Conclusion This method of streptokinase purification is superior to the previous conventional methods. PMID:23408770

  13. Proteome-scale purification of human proteins from bacteria

    PubMed Central

    Braun, Pascal; Hu, Yanhui; Shen, Binghua; Halleck, Allison; Koundinya, Malvika; Harlow, Ed; LaBaer, Joshua

    2002-01-01

    The completion of the human genome project and the development of high-throughput approaches herald a dramatic acceleration in the pace of biological research. One of the most compelling next steps will be learning the functional roles of all proteins. Achievement of this goal depends in part on the rapid expression and isolation of proteins at large scale. We exploited recombinational cloning to facilitate the development of methods for the high-throughput purification of human proteins. cDNAs were introduced into a master vector from which they could be rapidly transferred into a variety of protein expression vectors for further analysis. A test set of 32 sequence-verified human cDNAs of various sizes and activities was moved into four different expression vectors encoding different affinity-purification tags. By means of an automatable 2-hr protein purification procedure, all 128 proteins were purified and subsequently characterized for yield, purity, and steps at which losses occurred. Under denaturing conditions when the His6 tag was used, 84% of samples were purified. Under nondenaturing conditions, both the glutathione S-transferase and maltose-binding protein tags were successful in 81% of samples. The developed methods were applied to a larger set of 336 randomly selected cDNAs. Sixty percent of these proteins were successfully purified under denaturing conditions and 82% of these under nondenaturing conditions. A relational database, FLEXProt, was built to compare properties of proteins that were successfully purified and proteins that were not. We observed that some domains in the Pfam database were found almost exclusively in proteins that were successfully purified and thus may have predictive character. PMID:11880620

  14. Purification of C-phycocyanin from Spirulina (Arthrospira) fusiformis.

    PubMed

    Minkova, K M; Tchernov, A A; Tchorbadjieva, M I; Fournadjieva, S T; Antova, R E; Busheva, M Ch

    2003-04-10

    C-phycocyanin was purified from Spirulina (Arthrospira) fusiformis by a multi-step treatment of the crude extract with rivanol in a ratio 10:1 (v/v), followed by 40% saturation with ammonium sulfate. After removal of rivanol by gel-filtration on Sephadex G-25, the pigment solution was saturated to 70% with ammonium sulfate. After the last step of purification, C-phycocyanin had an emission and absorption maxima at 620 and 650 nm, respectively and absorbance ratio A(620)/A(280) of 4.3, which are specific for the pure biliprotein. Its homogeneity was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, yielding two bands of molecular masses 19500 and 21500 kDa, corresponding to alpha and beta subunits of the pigment, respectively. The yield of C-phycocyanin was approximately 46% from its content in the crude extract. PMID:12668314

  15. Potlining Additives

    SciTech Connect

    Rudolf Keller

    2004-08-10

    In this project, a concept to improve the performance of aluminum production cells by introducing potlining additives was examined and tested. Boron oxide was added to cathode blocks, and titanium was dissolved in the metal pool; this resulted in the formation of titanium diboride and caused the molten aluminum to wet the carbonaceous cathode surface. Such wetting reportedly leads to operational improvements and extended cell life. In addition, boron oxide suppresses cyanide formation. This final report presents and discusses the results of this project. Substantial economic benefits for the practical implementation of the technology are projected, especially for modern cells with graphitized blocks. For example, with an energy savings of about 5% and an increase in pot life from 1500 to 2500 days, a cost savings of $ 0.023 per pound of aluminum produced is projected for a 200 kA pot.

  16. Phosphazene additives

    SciTech Connect

    Harrup, Mason K; Rollins, Harry W

    2013-11-26

    An additive comprising a phosphazene compound that has at least two reactive functional groups and at least one capping functional group bonded to phosphorus atoms of the phosphazene compound. One of the at least two reactive functional groups is configured to react with cellulose and the other of the at least two reactive functional groups is configured to react with a resin, such as an amine resin of a polycarboxylic acid resin. The at least one capping functional group is selected from the group consisting of a short chain ether group, an alkoxy group, or an aryloxy group. Also disclosed are an additive-resin admixture, a method of treating a wood product, and a wood product.

  17. Mammalian cell production and purification of progenipoietin, a dual-agonist chimaeric haematopoietic growth factor.

    PubMed

    Wood, David C; Mathis, Karl J; Joy, William D; Minnerly, John C; Pegg, Lyle E; Welply, Joseph K

    2003-02-01

    One member of the progenipoietin (ProGP) family of engineered proteins, ProGP-2, is a chimaeric dual cytokine receptor agonist, expressed in mammalian cells, that stimulates both human fetal liver tyrosine kinase-3 (Flt3) and the granulocyte-colony-stimulating-factor (G-CSF) receptor. The production of ProGP-2 on a small and large scale using either anti-(Flt3 ligand) antibody-affinity chromatography, or a combination of (NH4)2SO4 fractionation, anion-exchange chromatography, hydrophobic-interaction chromatography and preparative reverse-phase chromatography is described. ProGP-2 was produced in hollow-fibre reactors containing stably transfected NS0 cells. The productivity of ProGP-2 was initially high, but was found to decrease 3-4-fold over time. When the yield of ProGP-2 decreased, the combination of three conventional chromatography steps was required to meet protein purity similar to that achieved by the anti-(Flt3 ligand) chromatography method. In addition, a protease activity was observed in conditioned media from the hollow-fibre reactors that resulted in increased degradation of ProGP-2 that was removed by hydrophobic-interaction chromatography at higher pH. Together the results demonstrated a method for production and purification of ProGP-2 for additional studies on its haematopoietic activity. PMID:12578549

  18. Partial purification of saccharifying and cell wall-hydrolyzing enzymes from malt in waste from beer fermentation broth.

    PubMed

    Khattak, Waleed Ahmad; Kang, Minkyung; Ul-Islam, Mazhar; Park, Joong Kon

    2013-06-01

    A number of hydrolyzing enzymes that are secreted from malt during brewing, including cell wall-hydrolyzing, saccharide-hydrolyzing, protein-degrading, lipid-hydrolyzing, and polyphenol and thiol-hydrolyzing enzymes, are expected to exist in an active form in waste from beer fermentation broth (WBFB). In this study, the existence of these enzymes was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, after which enzyme extract was partially purified through a series of purification steps. The hydrolyzing enzyme activity was then measured under various conditions at each purification step using carboxymethyl cellulose as a substrate. The best hydrolyzing activities of partially purified enzymes were found at pH 4.5 and 50 °C in a citrate buffer system. The enzymes showed highest thermal stability at 30 °C when exposed for prolonged time. As the temperature increased gradually from 25 to 70 °C, yeast cells in the chemically defined medium with enzyme extract lost their cell wall and viability earlier than those without enzyme extract. Cell wall degradation and the release of cell matrix into the culture media at elevated temperature (45-70 °C) in the presence of enzyme extract were monitored through microscopic pictures. Saccharification enzymes from malt were relatively more active in the original WBFB than supernatant and diluted sediments. The presence of hydrolyzing enzymes from malt in WBFB is expected to play a role in bioethanol production using simultaneous saccharification and fermentation without the need for additional enzymes, nutrients, or microbial cells via a cell-free enzyme system. PMID:23377262

  19. Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids.

    PubMed

    Akutsu, Tomoko; Kitayama, Tetsushi; Watanabe, Ken; Sakurada, Koichi

    2015-01-01

    Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the β-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples. PMID:25270217

  20. Steps in Behavior Modividation.

    ERIC Educational Resources Information Center

    Straughan, James H.; And Others

    James H. Straughan lists five steps for modifying target behavior and four steps for working with teachers using behavior modification. Grant Martin and Harold Kunzelmann then outline an instructional program for pinpointing and recording classroom behaviors. (JD)

  1. Selective catalytic two-step process for ethylene glycol from carbon monoxide

    PubMed Central

    Dong, Kaiwu; Elangovan, Saravanakumar; Sang, Rui; Spannenberg, Anke; Jackstell, Ralf; Junge, Kathrin; Li, Yuehui; Beller, Matthias

    2016-01-01

    Upgrading C1 chemicals (for example, CO, CO/H2, MeOH and CO2) with C–C bond formation is essential for the synthesis of bulk chemicals. In general, these industrially important processes (for example, Fischer Tropsch) proceed at drastic reaction conditions (>250 °C; high pressure) and suffer from low selectivity, which makes high capital investment necessary and requires additional purifications. Here, a different strategy for the preparation of ethylene glycol (EG) via initial oxidative coupling and subsequent reduction is presented. Separating coupling and reduction steps allows for a completely selective formation of EG (99%) from CO. This two-step catalytic procedure makes use of a Pd-catalysed oxycarbonylation of amines to oxamides at room temperature (RT) and subsequent Ru- or Fe-catalysed hydrogenation to EG. Notably, in the first step the required amines can be efficiently reused. The presented stepwise oxamide-mediated coupling provides the basis for a new strategy for selective upgrading of C1 chemicals. PMID:27377550

  2. Selective catalytic two-step process for ethylene glycol from carbon monoxide.

    PubMed

    Dong, Kaiwu; Elangovan, Saravanakumar; Sang, Rui; Spannenberg, Anke; Jackstell, Ralf; Junge, Kathrin; Li, Yuehui; Beller, Matthias

    2016-01-01

    Upgrading C1 chemicals (for example, CO, CO/H2, MeOH and CO2) with C-C bond formation is essential for the synthesis of bulk chemicals. In general, these industrially important processes (for example, Fischer Tropsch) proceed at drastic reaction conditions (>250 °C; high pressure) and suffer from low selectivity, which makes high capital investment necessary and requires additional purifications. Here, a different strategy for the preparation of ethylene glycol (EG) via initial oxidative coupling and subsequent reduction is presented. Separating coupling and reduction steps allows for a completely selective formation of EG (99%) from CO. This two-step catalytic procedure makes use of a Pd-catalysed oxycarbonylation of amines to oxamides at room temperature (RT) and subsequent Ru- or Fe-catalysed hydrogenation to EG. Notably, in the first step the required amines can be efficiently reused. The presented stepwise oxamide-mediated coupling provides the basis for a new strategy for selective upgrading of C1 chemicals. PMID:27377550

  3. Diversity and dynamics of microbial communities at each step of treatment plant for potable water generation.

    PubMed

    Lin, Wenfang; Yu, Zhisheng; Zhang, Hongxun; Thompson, Ian P

    2014-04-01

    The dynamics of bacterial and eukaryotic community associated with each step of a water purification plant in China was investigated using 454 pyrosequencing and qPCR based approaches. Analysis of pyrosequencing revealed that a high degree diversity of bacterial and eukaryotic communities is present in the drinking water treatment process before sand filtration. In addition, the microbial compositions of the biofilm in the sand filters and those of the water of the putatively clear tanks were distinct, suggesting that sand filtration and chlorination treatments played primary roles in removing exposed microbial communities. Potential pathogens including Acinetobacter, Clostridium, Legionella, and Mycobacterium, co-occurred with protozoa such as Rhizopoda (Hartmannellidae), and fungi such as Penicillium and Aspergillus. Furthermore, this study supported the ideas based on molecular level that biofilm communities were different from those in corresponding water samples, and that the concentrations of Mycobacterium spp., Legionella spp., and Naegleria spp. in the water samples declined with each step of the water treatment process by qPCR. Overall, this study provides the first detailed evaluation of bacterial and eukaryotic diversity at each step of an individual potable water treatment process located in China. PMID:24268295

  4. A novel aqueous two phase system composed of a thermo-separating polymer and an organic solvent for purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus) peel.

    PubMed

    Amid, Mehrnoush; Manap, Yazid; Zohdi, Nor Khanani

    2014-01-01

    The purification of thermo-acidic amylase enzyme from red pitaya (Hylocereus polyrhizus) peel for the first time was investigated using a novel aqueous two-phase system (ATPS) consisting of a thermo-separating copolymer and an organic solvent. The effectiveness of different parameters such as molecular weight of the thermo-separating ethylene oxide-propylene oxide (EOPO) copolymer and type and concentration of organic solvent on the partitioning behavior of amylase was investigated. In addition, the effects of phase components, volume ratio (VR), pH and crude load of purification factor and yield of amylase were evaluated to achieve the optimum partition conditions of the enzyme. In the novel ATPS method, the enzyme was satisfactorily partitioned into the polymer-rich top phase in the system composed of 30% (w/w) EOPO 2500 and 15% (w/w) 2-propanol, at a volume ratio of 1.94 and with a crude load scale of 25% (w/w) at pH 5.0. Recovery and recycling of components was also measured in each successive step of the ATPS process. The enzyme was successfully recovered by the method with a high purification factor of 14.3 and yield of 96.6% and copolymer was also recovered and recycled at a rate above 97%, making the method was more economical than the traditional ATPS method. PMID:24858097

  5. Stepped Hydraulic Geometry in Stepped Channels

    NASA Astrophysics Data System (ADS)

    Comiti, F.; Cadol, D. D.; Wohl, E.

    2007-12-01

    Steep mountain streams typically present a stepped longitudinal profile. Such stepped channels feature tumbling flow, where hydraulic jumps represent an important source of channel roughness (spill resistance). However, the extent to which spill resistance persists up to high flows has not been ascertained yet, such that a faster, skimming flow has been envisaged to begin at those conditions. In order to analyze the relationship between flow resistance and bed morphology, a mobile bed physical model was developed at Colorado State University (Fort Collins, USA). An 8 m-long, 0.6 m-wide flume tilted at a constant 14% slope was used, testing 2 grain-size mixtures differing only for the largest fraction. Experiments were conducted under clear water conditions. Reach-averaged flow velocity was measured using salt tracers, bed morphology and flow depth by a point gage, and surface grain size using commercial image-analysis software. Starting from an initial plane bed, progressively higher flow rates were used to create different bed structures. After each bed morphology was stable with its forming discharge, lower-than-forming flows were run to build a hydraulic geometry curve. Results show that even though equilibrium slopes ranged from 8.5% to 14%, the reach-averaged flow was always sub-critical. Steps formed through a variety of mechanisms, with immobile clasts playing a dominant role by causing local scouring and/or trapping moving smaller particles. Overall, step height, step pool steepness, relative pool area and volume increased with discharge up to the threshold when the bed approached fully- mobilized conditions. For bed morphologies surpassing a minimum profile roughness, a stepped velocity- discharge relationship is evident, with sharp rises in velocity correlated with the disappearance of rollers in pools at flows approaching the formative discharge for each morphology. Flow resistance exhibits an opposite pattern, with drops in resistance being a function

  6. Hydrocarbon fuel additive

    SciTech Connect

    Ambrogio, S.

    1989-02-28

    This patent describes the method of fuel storage or combustion, wherein the fuel supply contains small amounts of water, the step of adding to the fuel supply an additive comprising a blend of a hydrophilic agent chosen from the group of ethylene glycol, n-butyl alcohol, and cellosolve in the range of 22-37% by weight; ethoxylated nonylphenol in the range of 26-35% by weight; nonylphenol polyethylene glycol ether in the range of 32-43% by weight.

  7. One Step to Learning.

    ERIC Educational Resources Information Center

    Thornton, Carol A.; And Others

    1980-01-01

    Described are activities and games incorporating a technique of "one step" which is used with children with learning difficulties. The purpose of "one step" is twofold, to minimize difficulties with typical trouble spots and to keep the step size of the instruction small. (Author/TG)

  8. A Step Circuit Program.

    ERIC Educational Resources Information Center

    Herman, Susan

    1995-01-01

    Aerobics instructors can use step aerobics to motivate students. One creative method is to add the step to the circuit workout. By incorporating the step, aerobic instructors can accommodate various fitness levels. The article explains necessary equipment and procedures, describing sample stations for cardiorespiratory fitness, muscular strength,…

  9. Optimization of water treatment methods for the purification of peat extraction derived runoff: Evaluation of chemical treatment response to variations in incoming water quality using a 2k factorial test design

    NASA Astrophysics Data System (ADS)

    Heiderscheidt, Elisangela; Ronkanen, Anna-Kaisa; Klöve, Björn

    2013-04-01

    The sustainable use of peatland areas requires measures to minimize and when possible eradicate the identified environmental impacts. The drainage of peatlands and other peat extraction, agriculture and forestry activities are known to increase the leaching of pollutant substances resulting in the eutrophication and siltation of receiving water bodies, causing water quality deterioration. Due to the geochemistry characteristics of peat soils the quality of peatland derived runoff water is known to oscillate with location and also with variations in runoff and peak discharge occurrences. Affordable, simple and reliable purification methods that can purify waters rich in particulates, nutrients and dissolved organic carbon while capable of coping with incoming water quality variations are therefore required. Chemical treatment is considered one of the best available technologies for the purification of peat extraction runoff water in Finland; however, until recently little research had been applied on the development of this treatment method for the purification of non-point source pollution. Chemical purification, using metal salts as coagulant agents, is currently applied in several treatment facilities in Finnish peat extraction sites. Nevertheless, variations in runoff water quality and the lack of development of field process parameters has led to the application of high chemical dosages, significant and undesirable fluctuations in purification efficiency and high metal concentration in the discharging waters. This work aims to develop and optimize the chemical purification method by using high level scientific methods to evaluate the response of the purification process to variations in water quality which are typical of peatland derived runoff. The evaluation of how the purification process responds to these variations is a critical step which will enable the development of preventive measures and optimization of relevant process parameters and thus reduce the

  10. Radiolabeling of lipo-chitooligosaccharides using the NodH sulfotransferase: a two-step enzymatic procedure

    PubMed Central

    Gressent, Frédéric; Cullimore, Julie V; Ranjeva, Raoul; Bono, Jean-Jacques

    2004-01-01

    Background The NodH sulfotransferase from Sinorhizobium meliloti has been used to radiolabel lipochitooligosaccharidic (LCO) Nod factor signals with 35S from inorganic sulfate in a two-step enzymatic procedure. The first step involved the production of 3'-phosphoadenosine 5'-phosphosulfate (PAPS), a sulphate donor, using enzymes contained in a yeast extract, and the second step used the NodH enzyme. However with this established procedure, only a low incorporation of the initial inorganic sulfate into the Nod factors was obtained (about 7% after purification of the labeled compounds). The aim of this work was to optimize the radiolabelling of Nod factors with 35S. Results The limiting step has been shown to be the sulfation of ATP and its subsequent conversion into PAPS (first step), the sulfate donor for the NodH sulfotransferase activity (second step). By the addition of GTP to the reaction mixture and by manipulating the [ATP]/[Mg2+] ratio the yield of PAPS has been increased from 13% to 80%. Using the radiolabeled PAPS we have shown that the efficiency of sulfate transfer to LCOs, by the recombinant S. meliloti NodH sulfotransferase is strongly influenced by the length of the oligosaccharide chain. Variations in the substitutions on the non-reducing sugar, including the structure of the fatty acyl chain, had little effect and Nod factors from the heterologous bacterium Rhizobium tropici could be sulfated by NodH from S. meliloti. Conclusions By characterizing the two steps we have optimized the procedure to radiolabel biologically-important, lipo-chitooligosaccharide (LCO) Nod factors to a specific radioactivity of about 800 Ci.mmol-1 with an incorporation of 60% of the initial inorganic sulfate. The two-step sulfation procedure may be used to radiolabel a variety of related LCO molecules. PMID:15084228

  11. Soft-Bake Purification of SWCNTs Produced by Pulsed Laser Vaporization

    NASA Technical Reports Server (NTRS)

    Yowell, Leonard; Nikolaev, Pavel; Gorelik, Olga; Allada, Rama Kumar; Sosa, Edward; Arepalli, Sivaram

    2013-01-01

    The "soft-bake" method is a simple and reliable initial purification step first proposed by researchers at Rice University for single-walled carbon nanotubes (SWCNT) produced by high-pressure carbon mon oxide disproportionation (HiPco). Soft-baking consists of annealing as-produced (raw) SWCNT, at low temperatures in humid air, in order to degrade the heavy graphitic shells that surround metal particle impurities. Once these shells are cracked open by the expansion and slow oxidation of the metal particles, the metal impurities can be digested through treatment with hydrochloric acid. The soft-baking of SWCNT produced by pulsed-laser vaporization (PLV) is not straightforward, because the larger average SWCNT diameters (.1.4 nm) and heavier graphitic shells surrounding metal particles call for increased temperatures during soft-bake. A part of the technology development focused on optimizing the temperature so that effective cracking of the graphitic shells is balanced with maintaining a reasonable yield, which was a critical aspect of this study. Once the ideal temperature was determined, a number of samples of raw SWCNT were purified using the soft-bake method. An important benefit to this process is the reduced time and effort required for soft-bake versus the standard purification route for SWCNT. The total time spent purifying samples by soft-bake is one week per batch, which equates to a factor of three reduction in the time required for purification as compared to the standard acid purification method. Reduction of the number of steps also appears to be an important factor in improving reproducibility of yield and purity of SWCNT, as small deviations are likely to get amplified over the course of a complicated multi-step purification process.

  12. Purification of the proprotein convertase furin by affinity chromatography based on PC-specific inhibitors

    PubMed Central

    Kuester, Miriam; Becker, Gero L.; Hardes, Kornelia; Lindberg, Iris; Steinmetzer, Torsten; Than, Manuel E.

    2013-01-01

    In eucaryotes, many secreted proteins and peptides are proteolytically excised from larger precursor proteins by a specific class of serine proteases, the proprotein/prohormone convertases (PCs). This cleavage is essential for substrate activation, making the PCs very interesting pharmacological targets in cancer and infectious disease research. Correspondingly, their structure, function and inhibition are intensely studied – studies that require the respective target proteins in large amounts and at high purity. Here we describe the development of a novel purification protocol of furin, the best-studied member of the PC family. We combined the heterologous expression of furin from CHO cells with a novel purification scheme employing an affinity step that efficiently extracts only active furin from the conditioned medium by using furin-specific inhibitor moieties as bait. Several potential affinity tags were synthesized and their binding to furin characterized. The best compound, Biotin-(Adoa)2-Arg-Pro-Arg-4-Amba coupled to streptavidin-Sepharose beads, was used in a three-step chromatographic protocol and routinely resulted in a high yield of a homogeneous furin preparation with a specific activity of ~60 units/mg protein. This purification and the general strategy can easily be adapted to the efficient purification of other PC family members. PMID:21875402

  13. Neuregulin: First Steps Towards a Structure

    NASA Technical Reports Server (NTRS)

    Ferree, D. S.; Malone, C. C.; Karr, L. J.

    2003-01-01

    Neuregulins are growth factor domain proteins with diverse bioactivities, such as cell proliferation, receptor binding, and differentiation. Neureguh- 1 binds to two members of the ErbB class I tyrosine kinase receptors, ErbB3 and ErbB4. A number of human cancers overexpress the ErbB receptors, and neuregulin can modulate the growth of certain cancer types. Neuregulin-1 has been shown to promote the migration of invasive gliomas of the central nervous system. Neuregulin has also been implicated in schizophrenia, multiple sclerosis and abortive cardiac abnormalities. The full function of neuregulin-1 is not known. In this study we are inserting a cDNA clone obtained from American Type Culture Collection into E.coli expression vectors to express neuregulin- 1 protein. Metal chelate affinity chromatography is used for recombinant protein purification. Crystallization screening will proceed for X-ray diffraction studies following expression, optimization, and protein purification. In spite of medical and scholarly interest in the neuregulins, there are currently no high-resolution structures available for these proteins. Here we present the first steps toward attaining a high-resolution structure of neuregulin- 1, which will help enable us to better understand its function

  14. A heme fusion tag for protein affinity purification and quantification

    PubMed Central

    Asher, Wesley B; Bren, Kara L

    2010-01-01

    We report a novel affinity-based purification method for proteins expressed in Escherichia coli that uses the coordination of a heme tag to an l-histidine-immobilized sepharose (HIS) resin. This approach provides an affinity purification tag visible to the eye, facilitating tracking of the protein. We show that azurin and maltose binding protein are readily purified from cell lysate using the heme tag and HIS resin. Mild conditions are used; heme-tagged proteins are bound to the HIS resin in phosphate buffer, pH 7.0, and eluted by adding 200–500 mM imidazole or binding buffer at pH 5 or 8. The HIS resin exhibits a low level of nonspecific binding of untagged cellular proteins for the systems studied here. An additional advantage of the heme tag-HIS method for purification is that the heme tag can be used for protein quantification by using the pyridine hemochrome absorbance method for heme concentration determination. PMID:20665691

  15. Matching relations for optimal entanglement concentration and purification

    PubMed Central

    Kong, Fan-Zhen; Xia, Hui-Zhi; Yang, Ming; Yang, Qing; Cao, Zhuo-Liang

    2016-01-01

    The bilateral controlled NOT (CNOT) operation plays a key role in standard entanglement purification process, but the CNOT operation may not be the optimal joint operation in the sense that the output entanglement is maximized. In this paper, the CNOT operations in both the Schmidt-projection based entanglement concentration and the entanglement purification schemes are replaced with a general joint unitary operation, and the optimal matching relations between the entangling power of the joint unitary operation and the non-maximal entangled channel are found for optimizing the entanglement in- crement or the output entanglement. The result is somewhat counter-intuitive for entanglement concentration. The output entanglement is maximized when the entangling power of the joint unitary operation and the quantum channel satisfy certain relation. There exist a variety of joint operations with non-maximal entangling power that can induce a maximal output entanglement, which will greatly broaden the set of the potential joint operations in entanglement concentration. In addition, the entanglement increment in purification process is maximized only by the joint unitary operations (including CNOT) with maximal entangling power. PMID:27189800

  16. Matching relations for optimal entanglement concentration and purification.

    PubMed

    Kong, Fan-Zhen; Xia, Hui-Zhi; Yang, Ming; Yang, Qing; Cao, Zhuo-Liang

    2016-01-01

    The bilateral controlled NOT (CNOT) operation plays a key role in standard entanglement purification process, but the CNOT operation may not be the optimal joint operation in the sense that the output entanglement is maximized. In this paper, the CNOT operations in both the Schmidt-projection based entanglement concentration and the entanglement purification schemes are replaced with a general joint unitary operation, and the optimal matching relations between the entangling power of the joint unitary operation and the non-maximal entangled channel are found for optimizing the entanglement in- crement or the output entanglement. The result is somewhat counter-intuitive for entanglement concentration. The output entanglement is maximized when the entangling power of the joint unitary operation and the quantum channel satisfy certain relation. There exist a variety of joint operations with non-maximal entangling power that can induce a maximal output entanglement, which will greatly broaden the set of the potential joint operations in entanglement concentration. In addition, the entanglement increment in purification process is maximized only by the joint unitary operations (including CNOT) with maximal entangling power. PMID:27189800

  17. STEP: A Futurevision, Today

    NASA Technical Reports Server (NTRS)

    1994-01-01

    STEP (STandard for the Exchange of Product Model Data) is an innovative software tool that allows the exchange of data between different programming systems to occur and helps speed up the designing in various process industries. This exchange occurs easily between those companies that have STEP, and many industries and government agencies are requiring that their vendors utilize STEP in their computer aided design projects, such as in the areas of mechanical, aeronautical, and electrical engineering. STEP allows the process of concurrent engineering to occur and increases the quality of the design product. One example of the STEP program is the Boeing 777, the first paperless airplane.

  18. Purification of human recombinant interleukin 1 receptor antagonist proteins upon Bacillus subtilis sporulation.

    PubMed

    Maurizi, G; Di Cioccio, V; Macchia, G; Bossù, P; Bizzarri, C; Visconti, U; Boraschi, D; Tagliabue, A; Ruggiero, P

    1997-03-01

    Human interleukin 1 receptor antagonist (IL-1ra) and IL-1ra mutants were constitutively expressed in recombinant Bacillus subtilis in endocellular and active form. In order to optimize the purification of the recombinant proteins, a new method has been developed. After bacterial growth in fermenter, release of recombinant protein was achieved by starvation-induced sporulation. The sporulation supernatant was recovered by centrifugation, filtered, and subjected sequentially to cation- and anion-exchange chromatography. Alternatively, the fermenter's contents were directly subjected to expanded bed adsorption on a Streamline cation-exchange column, thus avoiding the centrifugation and filtration steps. Up to 88 mg of biological active purified recombinant protein per liter of culture was obtained, with a 72-79% recovery and 98% purity, depending on the molecule. By using the method described here, it is possible to achieve a spontaneous release of recombinant proteins expressed endocellularly at high levels in B. subtilis without need of a cell breakage step. Thus, this method could allow purification of the endocellular recombinant protein as if it were secreted. Furthermore, when using the expanded bed adsorption, highly purified protein was obtained in only two steps after sporulation. Among the advantages of the method, one of the most relevant is the possibility of keeping the system closed up to completion of the first purification step. PMID:9056487

  19. Hydrogen production and purification for fuel cell applications

    NASA Astrophysics Data System (ADS)

    Chin, Soo Yin

    The increased utilization of proton-exchange membrane (PEM) fuel cells as an alternative to internal combustion engines is expected to increase the demand for hydrogen, which is used as the energy source in these systems. Currently, production of hydrogen for fuel cells is primarily achieved via steam reforming, partial oxidation or autothermal reforming of natural gas, or steam reforming of methanol. However, in all of these processes CO is a by-product that must be subsequently removed due to its adverse effects on the Pt-based electrocatalysts of the PEM fuel cell. Our efforts have focused on production of CO-free hydrogen via catalytic decomposition of hydrocarbons and purification of H2 via the preferential oxidation of CO. The catalytic decomposition of hydrocarbons is an attractive alternative for the production of H2. Previous studies utilizing methane have shown that this approach can indeed produce CO-free hydrogen, with filamentous carbon formed as the by-product and deposited on the catalyst. We have further extended this approach to the decomposition of ethane. In addition to hydrogen and filamentous carbon however, methane is also formed in this case as a by-product. Studies conducted at different temperatures and space velocities suggest that hydrogen is the primary product while methane is formed in a secondary step. Ni/SiO2 catalysts are active for ethane decomposition at temperatures above 500°C. Although the yield of hydrogen increases with temperature, the catalyst deactivation rate also accelerates at higher temperatures. The preferential oxidation of CO is currently used for the purification of CO-contaminated hydrogen streams due to its efficiency and simplicity. Conventional Pt catalysts used for this reaction have been shown to effectively remove CO, but have limited selectivity (i.e., substantial amounts of H 2 also react with O2). Our work focused on alternative catalytic materials, such as Ru and bimetallic Ru-based catalysts (Pt-Ru, Ru

  20. Doing That Thing That Scientists Do: A Discovery-Driven Module on Protein Purification and Characterization for the Undergraduate Biochemistry Laboratory Classroom

    ERIC Educational Resources Information Center

    Garrett, Teresa A.; Osmundson, Joseph; Isaacson, Marisa; Herrera, Jennifer

    2015-01-01

    In traditional introductory biochemistry laboratory classes students learn techniques for protein purification and analysis by following provided, established, step-by-step procedures. Students are exposed to a variety of biochemical techniques but are often not developing procedures or collecting new, original data. In this laboratory module,…

  1. Dynamic mechanical analysis of hydrogen purification substrates and membranes

    NASA Astrophysics Data System (ADS)

    Steinborn, Brandon

    Porous 420 stainless steel hydrogen purification substrates were fabricated using an ExOne R2 printer and sintered at temperatures of 1075 °C and 1100 °C for times ranging from 15 minutes to 240 minutes. Coatings of 1 micron silica beads, silica sol-gel, and palladium were applied to the sintered structure. Mechanical properties/degradation of each substrate/coating combination were evaluated using a cyclic 3-point loading condition imposed by a TA Q800 dynamic mechanical analysis unit (DMA). A constant deformation procedure was used while the required drive force for deformation and the elasticity (tan delta) were recorded throughout the cycle. Findings with respect to coating additions include: drive force increases with the addition of each coating, tan delta decreases with ceramic additions and increases with palladium addition (eventually decreases when membrane fails), and tan delta values become comparable with the addition of palladium regardless of other parameters. Findings with respect to sintering time and temperature include: drive force increases with increased sintering time and temperature, tan delta increases with increased sintering time at 1075 °C, and tan delta decreases with increased sintering time at 1100 °C. Overall, the palladium layer would likely remain intact in service due to actual force oscillations not being as extreme in service, poisoning would likely be the life limiting factor. Keywords: Sintering, dynamic mechanical properties, porous stainless steel, hydrogen purification, sol-gel.

  2. Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

    NASA Astrophysics Data System (ADS)

    Bitner, Rex M.; Koller, Susan C.

    2002-06-01

    The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

  3. Cyclic steps on ice

    NASA Astrophysics Data System (ADS)

    Yokokawa, M.; Izumi, N.; Naito, K.; Parker, G.; Yamada, T.; Greve, R.

    2016-05-01

    Boundary waves often form at the interface between ice and fluid flowing adjacent to it, such as ripples under river ice covers, and steps on the bed of supraglacial meltwater channels. They may also be formed by wind, such as the megadunes on the Antarctic ice sheet. Spiral troughs on the polar ice caps of Mars have been interpreted to be cyclic steps formed by katabatic wind blowing over ice. Cyclic steps are relatives of upstream-migrating antidunes. Cyclic step formation on ice is not only a mechanical but also a thermodynamic process. There have been very few studies on the formation of either cyclic steps or upstream-migrating antidunes on ice. In this study, we performed flume experiments to reproduce cyclic steps on ice by flowing water, and found that trains of steps form when the Froude number is larger than unity. The features of those steps allow them to be identified as ice-bed analogs of cyclic steps in alluvial and bedrock rivers. We performed a linear stability analysis and obtained a physical explanation of the formation of upstream-migrating antidunes, i.e., precursors of cyclic steps. We compared the results of experiments with the predictions of the analysis and found the observed steps fall in the range where the analysis predicts interfacial instability. We also found that short antidune-like undulations formed as a precursor to the appearance of well-defined steps. This fact suggests that such antidune-like undulations correspond to the instability predicted by the analysis and are precursors of cyclic steps.

  4. The Isolation of Invertase from Baker's Yeast: A Four-Part Exercise in Protein Purification and Characterization

    ERIC Educational Resources Information Center

    Timerman, Anthony P.; Fenrick, Angela M.; Zamis, Thomas M.

    2009-01-01

    A sequence of exercises for the isolation and characterization of invertase (E.C. 3.1.2.26) from baker's yeast obtained from a local grocery store is outlined. Because the enzyme is colorless, the use of colored markers and the sequence of purification steps are designed to "visualize" the process by which a colorless protein is selectively…

  5. Reverse osmosis water purification system

    NASA Technical Reports Server (NTRS)

    Ahlstrom, H. G.; Hames, P. S.; Menninger, F. J.

    1986-01-01

    A reverse osmosis water purification system, which uses a programmable controller (PC) as the control system, was designed and built to maintain the cleanliness and level of water for various systems of a 64-m antenna. The installation operates with other equipment of the antenna at the Goldstone Deep Space Communication Complex. The reverse osmosis system was designed to be fully automatic; with the PC, many complex sequential and timed logic networks were easily implemented and are modified. The PC monitors water levels, pressures, flows, control panel requests, and set points on analog meters; with this information various processes are initiated, monitored, modified, halted, or eliminated as required by the equipment being supplied pure water.

  6. The Stepping Stone Approach

    NASA Astrophysics Data System (ADS)

    Brumfitt, A.

    Education is a profession in its own right. It has its own parameters, passions and language. Having the responsibility both of educare and educere, education has a focus of delivering specific factual knowledge whilst drawing out the creative mind. Space Science is a special vehicle having the properties of both educare and educere. It has a magic and wonder that touches the very essence of an individual and his place in time and space; it offers the "wow" factor that all teachers strive for. Space Science is the wrapping paper for other elements in the curriculum, e.g. cross-curricula and skill-based activities, such as language development, creativity, etc. as well as the pure sciences which comprise of engineering, physics and other natural sciences from astronomy to chemistry to biology. Each of these spheres of influence are relevant from kindergarten to undergraduate studies and complement, and in addition support informal education in museums, science centers and the world of e-learning. ESA Science Education has devised the "Stepping Stone Approach" to maximize the greatest outreach to all education stakeholders in Europe. In this paper we illustrate how to best reach these target groups with very specific activities to trigger and sustain enthusiasm whilst supporting the pedagogical, subject content and skill-based needs of a prescribed curriculum.

  7. Liquid membrane purification of biogas

    SciTech Connect

    Majumdar, S.; Guha, A.K.; Lee, Y.T.; Papadopoulos, T.; Khare, S. . Dept. of Chemistry and Chemical Engineering)

    1991-03-01

    Conventional gas purification technologies are highly energy intensive. They are not suitable for economic removal of CO{sub 2} from methane obtained in biogas due to the small scale of gas production. Membrane separation techniques on the other hand are ideally suited for low gas production rate applications due to their modular nature. Although liquid membranes possess a high species permeability and selectivity, they have not been used for industrial applications due to the problems of membrane stability, membrane flooding and poor operational flexibility, etc. A new hollow-fiber-contained liquid membrane (HFCLM) technique has been developed recently. This technique overcomes the shortcomings of the traditional immobilized liquid membrane technology. A new technique uses two sets of hydrophobic, microporous hollow fine fibers, packed tightly in a permeator shell. The inter-fiber space is filled with an aqueous liquid acting as the membrane. The feed gas mixture is separated by selective permeation of a species through the liquid from one fiber set to the other. The second fiber set carries a sweep stream, gas or liquid, or simply the permeated gas stream. The objectives (which were met) of the present investigation were as follows. To study the selective removal of CO{sub 2} from a model biogas mixture containing 40% CO{sub 2} (the rest being N{sub 2} or CH{sub 4}) using a HFCLM permeator under various operating modes that include sweep gas, sweep liquid, vacuum and conventional permeation; to develop a mathematical model for each mode of operation; to build a large-scale purification loop and large-scale permeators for model biogas separation and to show stable performance over a period of one month.

  8. Purification of bovine thyroid-stimulating hormone by a monoclonal antibody

    SciTech Connect

    Lock, A.J.; van Denderen, J.; Aarden, L.A.

    1988-01-01

    A monoclonal antibody directed against bovine TSH was obtained by hybridoma technology. This antibody was specific for TSH and did not react with bovine LH and FSH. Affinity chromatography of crude TSH was performed on anti-TSH Sepharose. Bovine TSH was purified in a single step to near homogeneity by this technique, as shown by cation exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified TSH. The biological activity of the hormone was not affected during the purification, as determined by (/sup 3/H)thymidine incorporation of the TSH-dependent FRTL5 cell line. The results indicate that affinity purification of TSH by means of a monoclonal antibody is a simple one-step procedure for the production of biologically active, highly purified TSH.

  9. Purification of very high density lipoproteins by differential density gradient ultracentrifugation.

    PubMed

    Haunerland, N H; Ryan, R O; Law, J H; Bowers, W S

    1987-03-01

    Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources. PMID:3578796

  10. Effectiveness of various processing steps for viral clearance of therapeutic proteins: database analyses of commonly used steps.

    PubMed

    Cipriano, Dana; Burnham, Michael; Hughes, Joseph V

    2012-01-01

    The successful implementation of any biologically derived product in human clinical trials and as a marketed biopharmaceutical requires the critical utilization of effective viral clearance steps. As biologic products have inherent risks of potentially carrying and or amplifying adventitious viruses that may be present in or introduced into the original materials, a number of processing steps are needed to provide adequate virus removal. Some common process steps are introduced into downstream purification schemes that provide a physical means to separate and/or remove viruses from the therapeutic protein. The physical steps often include virus-removing filters and chromatographic resins in column or membrane configurations, but can also include the introduction of irradiation, high heat steps, or other means for destroying the infectivity of a virus. Chemical treatment steps are often utilized as a means to inactivate a wide variety of virus types. A general overview is provided that describes the most commonly used techniques for virus removal or inactivation for the validation of virus clearance. Data sets from studies performed at WuXi AppTec for a wide variety of biologics reveal a number of steps that provide guidance for the design of process steps dedicated to viral clearance. The overall efficiency of several process steps reveals a number of efficient, robust steps, such as nanofiltration which can be designed for removal of almost all viral species. Exposure to a low pH or solvent detergent is also a robust step for inactivating enveloped virus. Steps with greater variances in predictability include chromatography steps such as capture columns and anion exchange resins. A lower removal capacity is typically expected for other chromatography steps such as cation exchange steps. PMID:22735960

  11. Tall fescue seed extraction and partial purification of ergot alkaloids

    NASA Astrophysics Data System (ADS)

    Bush, Lowell

    2014-12-01

    Many substances in the tall fescue/endophyte association (Schedonorus arundinaceus/Epichloë coenophiala) have biological activity. Of these compounds only the ergot alkaloids are known to have significant mammalian toxicity and the predominant ergot alkaloids are ergovaline and ergovalinine. Because synthetically produced ergovaline is difficult to obtain, we developed a seed extraction and partial purification protocol for ergovaline/ergovalinine that provided a biologically active product. Tall fescue seed was ground and packed into several different sized columns for liquid extraction. Smaller particle size and increased extraction time increased efficiency of extraction. Our largest column was a 114 × 52 × 61 cm (W×L×D) stainless steel tub. Approximately 150 kg of seed could be extracted in this tub. The extraction was done with 80% ethanol. When the solvent front migrated to bottom of the column, flow was stopped and seed was allowed to steep for at least 48 h. Light was excluded from the solvent from the beginning of this step to the end of the purification process. Following elution, ethanol was removed from the eluate by evaporation at room temperature. Resulting syrup was freeze-dried. About 80% recovery of alkaloids was achieved with 18-fold increase in concentration of ergovaline. Initial purification of the dried product was accomplished by extracting with hexane/water (6:1, v/v) and the hexane fraction was discarded. The aqueous fraction was extracted with chloroform, the aqueous layer discarded, after which the chloroform was removed with a resulting 20-fold increase of ergovaline. About 65% of the ergovaline was recovered from the chloroform residue for an overall recovery of 50%. The resultant partially purified ergovaline had biological activities in in vivo and in vitro bovine bioassays that approximate that of synthetic ergovaline.

  12. Purification of polymorphic components of complex genomes

    DOEpatents

    Stodolsky, Marvin

    1991-01-01

    A method is disclosed for processing related subject and reference macromolecule populations composed of complementary strands into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments.

  13. Purification of polymorphic components of complex genomes

    DOEpatents

    Stodolsky, M.

    1988-01-21

    A method for processing related subject and reference macromolecule composed of complementary strand into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments. 1 fig.

  14. Purification of polymorphic components of complex genomes

    DOEpatents

    Stodolsky, M.

    1991-07-16

    A method is disclosed for processing related subject and reference macromolecule populations composed of complementary strands into their respective subject and reference populations of representative fragments and effectuating purification of unique polymorphic subject fragments. 1 figure.

  15. An improved method for purification of recombinant truncated heme oxygenase-1 by expanded bed adsorption and gel filtration.

    PubMed

    Hu, Hong-Bo; Wang, Wei; Han, Ling; Zhou, Wen-Pu; Zhang, Xue-Hong

    2007-03-01

    Recombinant truncated human heme oxygenase-1 (hHO-1) expressed in Escherichia coli was efficiently separated and purified from feedstock by DEAE-ion exchange expanded bed adsorption. Protocol optimization of hHO-1 on DEAE adsorbent resulted in adsorption in 0 M NaCl and elution in 150 mM NaCl at a pH of 8.5. The active enzyme fractions separated from the expanded bed column were further purified by a Superdex 75 gel filtration step. The specific hHO-1 activity increased from 0.82 +/- 0.05 to 24.8 +/- 1.8 U/mg during the whole purification steps. The recovery and purification factor of truncated hHO-1 of the whole purification were 72.7 +/- 4.7 and 30.2 +/- 2.3%, respectively. This purification process can decrease the demand on the preparation of feedstock and simplify the purification process. PMID:17160582

  16. Golgi-Cox Staining Step by Step

    PubMed Central

    Zaqout, Sami; Kaindl, Angela M.

    2016-01-01

    Golgi staining remains a key method to study neuronal morphology in vivo. Since most protocols delineating modifications of the original staining method lack details on critical steps, establishing this method in a laboratory can be time-consuming and frustrating. Here, we describe the Golgi-Cox staining in such detail that should turn the staining into an easily feasible method for all scientists working in the neuroscience field. PMID:27065817

  17. Entanglement purification of unknown quantum states

    SciTech Connect

    Brun, Todd A.; Caves, Carlton M.; Schack, Ru''diger

    2001-04-01

    A concern has been expressed that ''the Jaynes principle can produce fake entanglement'' [R. Horodecki , Phys. Rev. A 59, 1799 (1999)]. In this paper we discuss the general problem of distilling maximally entangled states from N copies of a bipartite quantum system about which only partial information is known, for instance, in the form of a given expectation value. We point out that there is indeed a problem with applying the Jaynes principle of maximum entropy to more than one copy of a system, but the nature of this problem is classical and was discussed extensively by Jaynes. Under the additional assumption that the state {rho}{sup (N)} of the N copies of the quantum system is exchangeable, one can write down a simple general expression for {rho}{sup (N)}. By measuring one or more of the subsystems, one can gain information and update the state estimate for the remaining subsystems with the quantum version of the Bayes rule. Using this rule, we show how to modify two standard entanglement purification protocols, one-way hashing and recurrence, so that they can be applied to exchangeable states. We thus give an explicit algorithm for distilling entanglement from an unknown or partially known quantum state.

  18. Ultrafine polysaccharide nanofibrous membranes for water purification.

    PubMed

    Ma, Hongyang; Burger, Christian; Hsiao, Benjamin S; Chu, Benjamin

    2011-04-11

    Ultrafine polysaccharide nanofibers (i.e., cellulose and chitin) with 5-10 nm diameters were employed as barrier layers in a new class of thin-film nanofibrous composite (TFNC) membranes for water purification. In addition to concentration, the viscosity of the polysaccharide nanofiber coating suspension was also found to be affected by the pH value and ionic strength. When compared with two commercial UF membranes (PAN10 and PAN400), 10-fold higher permeation flux with above 99.5% rejection ratio were achieved by using ultrafine cellulose nanofibers-based TFNC membranes for ultrafiltration of oil/water emulsions. The very high surface-to-volume ratio and negatively charged surface of cellulose nanofibers, which lead to a high virus adsorption capacity as verified by MS2 bacteriophage testing, offer further opportunities in drinking water applications. The low cost of raw cellulose/chitin materials, the environmentally friendly fabrication process, and the impressive high-flux performance indicate that such ultrafine polysaccharide nanofibers-based TFNC membranes can surpass conventional membrane systems in many different water applications. PMID:21341679

  19. PLUTONIUM PURIFICATION PROCESS EMPLOYING THORIUM PYROPHOSPHATE CARRIER

    DOEpatents

    King, E.L.

    1959-04-28

    The separation and purification of plutonium from the radioactive elements of lower atomic weight is described. The process of this invention comprises forming a 0.5 to 2 M aqueous acidffc solution containing plutonium fons in the tetravalent state and elements with which it is normally contaminated in neutron irradiated uranium, treating the solution with a double thorium compound and a soluble pyrophosphate compound (Na/sub 4/P/sub 2/O/sub 7/) whereby a carrier precipitate of thorium A method is presented of reducing neptunium and - trite is advantageous since it destroys any hydrazine f so that they can be removed from solutions in which they are contained is described. In the carrier precipitation process for the separation of plutonium from uranium and fission products including zirconium and columbium, the precipitated blsmuth phosphate carries some zirconium, columbium, and uranium impurities. According to the invention such impurities can be complexed and removed by dissolving the contaminated carrier precipitate in 10M nitric acid, followed by addition of fluosilicic acid to about 1M, diluting the solution to about 1M in nitric acid, and then adding phosphoric acid to re-precipitate bismuth phosphate carrying plutonium.

  20. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, Robert K.; Bowman, Kenneth A.; Mazgaj, Robert M.; Cochran, C. Norman

    1983-10-25

    A method for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm.

  1. Process of electrolysis and fractional crystallization for aluminum purification

    DOEpatents

    Dawless, R.K.; Bowman, K.A.; Mazgaj, R.M.; Cochran, C.N.

    1983-10-25

    A method is described for purifying aluminum that contains impurities, the method including the step of introducing such aluminum containing impurities to a charging and melting chamber located in an electrolytic cell of the type having a porous diaphragm permeable by the electrolyte of the cell and impermeable to molten aluminum. The method includes further the steps of supplying impure aluminum from the chamber to the anode area of the cell and electrolytically transferring aluminum from the anode area to the cathode through the diaphragm while leaving impurities in the anode area, thereby purifying the aluminum introduced into the chamber. The method includes the further steps of collecting the purified aluminum at the cathode, and lowering the level of impurities concentrated in the anode area by subjecting molten aluminum and impurities in said chamber to a fractional crystallization treatment wherein eutectic-type impurities crystallize and precipitate out of the aluminum. The eutectic impurities that have crystallized are physically removed from the chamber. The aluminum in the chamber is now suited for further purification as provided in the above step of electrolytically transferring aluminum through the diaphragm. 2 figs.

  2. A fast and easy strategy for protein purification using “teabags”

    PubMed Central

    Castaldo, M.; Barlind, L.; Mauritzson, F.; Wan, P. T.; Snijder, H. J.

    2016-01-01

    Protein purification often involves affinity capture of proteins on stationary resin, alternatively proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment. PMID:27356497

  3. GABAB Receptor Constituents Revealed by Tandem Affinity Purification from Transgenic Mice*

    PubMed Central

    Bartoi, Tudor; Rigbolt, Kristoffer T. G.; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-01-01

    GABAB receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter γ-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABAB1, are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABAB receptors. The transgenic mice express GABAB1 isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABAB1 complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABAB receptors via the GABAB2 subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABAB2. The mice equipped with tags on GABAB1 facilitate validation and identification of native binding partners of GABAB receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  4. GABAB receptor constituents revealed by tandem affinity purification from transgenic mice.

    PubMed

    Bartoi, Tudor; Rigbolt, Kristoffer T G; Du, Dan; Köhr, Georg; Blagoev, Blagoy; Kornau, Hans-Christian

    2010-07-01

    GABA(B) receptors function as heterodimeric G-protein-coupled receptors for the neurotransmitter gamma-aminobutyric acid (GABA). Receptor subtypes, based on isoforms of the ligand-binding subunit GABA(B1), are thought to involve a differential set of associated proteins. Here, we describe two mouse lines that allow a straightforward biochemical isolation of GABA(B) receptors. The transgenic mice express GABA(B1) isoforms that contain sequences for a two-step affinity purification, in addition to their endogenous subunit repertoire. Comparative analyses of purified samples from the transgenic mice and wild-type control animals revealed two novel components of the GABA(B1) complex. One of the identified proteins, potassium channel tetramerization domain-containing protein 12, associates with heterodimeric GABA(B) receptors via the GABA(B2) subunit. In transfected hippocampal neurons, potassium channel tetramerization domain-containing protein 12 augmented axonal surface targeting of GABA(B2). The mice equipped with tags on GABA(B1) facilitate validation and identification of native binding partners of GABA(B) receptors, providing insight into the molecular mechanisms of synaptic modulation. PMID:20406808

  5. Sample purification for analysis of organochlorine pesticides in sediment and fish muscle.

    PubMed

    Pan, B; Liu, W X; Shi, Z; Cao, J; Shen, W R; Qing, B P; Sun, R; Tao, S

    2004-05-01

    The methods of sample purification for determinations of organochlorine pesticides (OCPs) in sediment and fish muscle were investigated in this study. A two-step procedure with preliminary cleanup and florisil column fraction was adopted. The working conditions of florisil column were firstly optimized. As to preliminary cleanup, liquid-liquid extraction by mixture of dimethyl formamide (DMF) and hexane and sulphonation by concentrated sulfuric acid were applied respectively and compared for the studied samples. The results indicated that liquid-liquid distribution could effectively separate lipids from fish muscle extracts, while failed in elimination of chlorophyll extracted from the relicts of hydrophytes in the sediment sample. In view of the sample appearance and clarity of gas chromatogram, sulphonation was more suitable to purify the sediment sample, however, the recoveries of the spiked standards were poor. The use of eluting solvent with stronger polarity could improve the corresponding recoveries significantly. Due to complete loss of Dieldrin, Endrin, and Endrin aldehyde after sulphonation, this pretreatment was improper to the three components. Sulfur, as the particular disturbing element in sediment, could be removed sufficiently by addition of activated copper powder. The relevant detection limits of the selected methods for OCPs in the studied substrates were also provided. PMID:15186026

  6. A general approach for the purification and quantitative glycomic analysis of human plasma.

    PubMed

    Tep, Samnang; Hincapie, Marina; Hancock, William S

    2012-03-01

    The development of a general method for the purification and quantitative glycomic analysis of human plasma samples to characterize global glycosylation changes shall be presented. The method involves multiple steps, including the depletion of plasma via multi-affinity chromatography to remove high abundant proteins, the enrichment of the lower abundant glycoproteins via multi-lectin affinity chromatography, the isotopic derivatization of released glycans, and quantitative analysis by MALDI-TOF MS. Isotopic derivatization of glycans is accomplished using the well-established chemistry of reductive amination to derivatize glycans with either a light analog ((12)C anthranilic acid) or a heavy analog ((13)C(7) anthranilic acid), which allows for the direct comparison of the alternately labeled glycans by MALDI-TOF MS. The method displays a tenfold linear dynamic range for both neutral and sialylated glycans with sub-picomolar sensitivity. Additionally, by using anthranilic acid, a very sensitive fluorophore, as the derivatization reagent, the glycans can be analyzed by chromatography with fluorescence detection. The utility of this methodology is highlighted by the many diseases and disorders that are known to either show or be the result of changes in glycosylation. A method that provides a generic approach for sample preparation and quantitative data will help to further advance the field of glycomics. PMID:22274286

  7. A comparison of protein A chromatographic stationary phases: performance characteristics for monoclonal antibody purification.

    PubMed

    Liu, Zhuo; Mostafa, Sigma S; Shukla, Abhinav A

    2015-01-01

    Protein A chromatography remains the dominant capture step used during the downstream purification of monoclonal antibodies (mAbs). With the recent expiry of the Repligen patent on recombinant Protein A, a variety of new Protein A resins have been introduced in the market. Given productivity limitations during downstream processing that have come into sharper focus with the recent increase in cell culture titers for mAbs, the selection of an appropriate Protein A resin has direct implications on the overall process economics of mAb production. The performance of seven different Protein A chromatographic resins was compared with respect to static binding capacity and dynamic binding capacity as a function of flow rate. This data was translated into a comparison of productivity (g mAb purified per unit resin volume per unit time) for the seven stationary phases. In addition, elution pH and host cell protein impurity levels after product capture on each of these resins were determined. The current article provides an effective methodology and dataset for the selection of the optimal Protein A chromatographic resin. PMID:24823474

  8. Phenylethynyl Containing Reactive Additives

    NASA Technical Reports Server (NTRS)

    Connell, John W. (Inventor); Smith, Joseph G., Jr. (Inventor); Hergenrother, Paul M. (Inventor)

    2002-01-01

    Phenylethynyl containing reactive additives were prepared from aromatic diamine, containing phenylethvnvl groups and various ratios of phthalic anhydride and 4-phenylethynviphthalic anhydride in glacial acetic acid to form the imide in one step or in N-methyl-2-pvrrolidinone to form the amide acid intermediate. The reactive additives were mixed in various amounts (10% to 90%) with oligomers containing either terminal or pendent phenylethynyl groups (or both) to reduce the melt viscosity and thereby enhance processability. Upon thermal cure, the additives react and become chemically incorporated into the matrix and effect an increase in crosslink density relative to that of the host resin. This resultant increase in crosslink density has advantageous consequences on the cured resin properties such as higher glass transition temperature and higher modulus as compared to that of the host resin.

  9. Design of ionic liquids for lipase purification.

    PubMed

    Ventura, Sónia P M; Sousa, Sílvia G; Freire, Mara G; Serafim, Luísa S; Lima, Alvaro S; Coutinho, João A P

    2011-09-15

    Aqueous two-phase systems (ATPS) are considered as efficient downstream processing techniques in the production and purification of enzymes, since they can be considered harmless to biomolecules due to their high water content and due to the possibility of maintaining a neutral pH value in the medium. A recent type of alternative ATPS is based on hydrophilic ionic liquids (ILs) and salting-out inducing salts. The aim of this work was to study the lipase (Candida antarctica lipase B - CaLB) partitioning in several ATPS composed of ionic liquids (ILs) and inorganic salts, and to identify the best IL for the enzyme purification. For that purpose a wide range of IL cations and anions, and some of their combinations were studied. For each system the enzyme partitioning between the two phases was measured and the purification factors and enzyme recoveries were determined. The results indicate that the lipase maximum purification and recovery were obtained for cations with a C(8) side alkyl chain, the [N(CN)(2)] anion and ILs belonging to the pyridinium family. However, the highest purification parameters were observed for 1-methyl-3-octylimidazolium chloride [C(8)mim]Cl, suggesting that the IL extraction capability does not result from a cumulative character of the individual characteristics of ILs. The results indicate that the IL based ATPS have an improved performance in the lipase purification and recovery. PMID:21852207

  10. Application of Eukaryotic Elongation Factor-2 Kinase (eEF-2K) for Cancer Therapy: Expression, Purification, and High-Throughput Inhibitor Screening.

    PubMed

    Tavares, Clint D J; Devkota, Ashwini K; Dalby, Kevin N; Cho, Eun Jeong

    2016-01-01

    Protein kinases have emerged as an important class of therapeutic targets, as they are known to be involved in pathological pathways linked to numerous human disorders. Major efforts to discover kinase inhibitors in both academia and pharmaceutical companies have centered on the development of robust assays and cost-effective approaches to isolate them. Drug discovery procedures often start with hit identification for lead development, by screening a library of chemicals using an appropriate assay in a high-throughput manner. Considering limitations unique to each assay technique and screening capability, intelligent integration of various assay schemes and level of throughput, in addition to the choice of chemical libraries, is the key to success of this initial step. Here, we describe the purification of the protein kinase, eEF-2K, and the utilization of three biochemical assays in the course of identifying small molecules that block its enzymatic reaction. PMID:26501899

  11. Color Addition and Subtraction Apps

    ERIC Educational Resources Information Center

    Ruiz, Frances; Ruiz, Michael J.

    2015-01-01

    Color addition and subtraction apps in HTML5 have been developed for students as an online hands-on experience so that they can more easily master principles introduced through traditional classroom demonstrations. The evolution of the additive RGB color model is traced through the early IBM color adapters so that students can proceed step by step…

  12. Color Addition and Subtraction Apps

    NASA Astrophysics Data System (ADS)

    Ruiz, Frances; Ruiz, Michael J.

    2015-10-01

    Color addition and subtraction apps in HTML5 have been developed for students as an online hands-on experience so that they can more easily master principles introduced through traditional classroom demonstrations. The evolution of the additive RGB color model is traced through the early IBM color adapters so that students can proceed step by step in understanding mathematical representations of RGB color. Finally, color addition and subtraction are presented for the X11 colors from web design to illustrate yet another real-life application of color mixing.

  13. The Borexino Solar Neutrino Experiment: Scintillator purification and surface contamination

    NASA Astrophysics Data System (ADS)

    Leung, Michael

    The Borexino Solar Neutrino Experiment will observe the monoenergetic (862 keV) 7Be neutrinos, produced in the solar reaction 7Be+e- →7 Li+nue. These neutrinos are the second most abundant species of solar neutrinos, with an expected flux at earth of 5 x 109/cm2/s. Using nu - e scattering in an aromatic liquid scintillator, Borexino will make the first real time measurement of the solar neutrino flux at energies less than 1 MeV. In addition to checking Standard Solar Model and neutrino oscillation predictions at low energies, Borexino will test the MSW vacuum-matter transition, luminosity constraint, and non-standard theories such as mass varying neutrinos. The Borexino detector will also be sensitive to supernova neutrinos, geoneutrinos, reactor neutrinos, and pep solar neutrinos. The pep measurement will tightly constrain the primary pp solar neutrino flux whose energy is below the Borexino threshold. With an expected rate of 35 events per day from solar 7Be neutrinos, the maximum tolerable background rate is one count per day. Removal of radioactive isotopes from the liquid scintillator is essential for the experiment's success and will be achieved with purification techniques including filtration, distillation, water extraction, nitrogen stripping, and silica gel adsorption. Results from small-scale purification efficiency tests are presented. Water extraction showed moderate but inadequate removal of 210Po which is a dominant background. Distillation reduced 210Po by a factor of more than 500. Online purification involves cycling over 300 m3 of scintillator from the detector though the purification plants. Flow patterns within the detector that influence the purification efficiency were determined with numerical simulations. Poor flow in the prototype Counting Test Facility showed effectively stagnant volumes within the detector. These are not present in the larger Borexino detector. Surface contamination in Borexino arises primarily from contact with

  14. Expression and purification of integral membrane metallopeptidase HtpX.

    PubMed

    Arolas, Joan L; García-Castellanos, Raquel; Goulas, Theodoros; Akiyama, Yoshinori; Gomis-Rüth, F Xavier

    2014-07-01

    Little is known about the catalytic mechanism of integral membrane (IM) peptidases. HtpX is an IM metallopeptidase that plays a central role in protein quality control by preventing the accumulation of misfolded proteins in the membrane. Here we report the recombinant overexpression and purification of a catalytically ablated form of HtpX from Escherichia coli. Several E. coli strains, expression vectors, detergents, and purification strategies were tested to achieve maximum yields of pure and well-folded protein. HtpX was successfully overexpressed in E. coli BL21(DE3) cells using a pET-derived vector attaching a C-terminal His8-tag, extracted from the membranes using octyl-β-d-glucoside, and purified to homogeneity in the presence of this detergent in three consecutive steps: cobalt-affinity, anion-exchange, and size-exclusion chromatography. The production of HtpX in milligram amounts paves the way for structural studies, which will be essential to understand the catalytic mechanism of this IM peptidase and related family members. PMID:24769134

  15. A hybrid solid-fluorous phase radioiodination and purification platform.

    PubMed

    Dzandzi, James P K; Vera, Denis R Beckford; Valliant, John F

    2014-07-01

    A new class of fluorous materials was developed to create a hybrid solid-solution phase strategy for the expedient preparation and HPLC-free purification of (125) I-labeled compounds. The system is referred to as a hybrid platform in that it combines solution phase labeling and fluorous solid-phase purification in one step as opposed to two separate individual processes. Treatment of fluorous arylstannanes coated on fluorous silica with [(125) I]NaI and the appropriate oxidant made it possible to produce and selectively isolate the nonfluorous radiolabeled products in high purity (>98%) free from excess starting material and unreacted radioiodine. Examples included simple aryl and heterocyclic (click) derivatives, known radiopharmaceuticals including meta-iodobenzylguanidine (MIBG) and iododeoxyuridine (IUdR), and a new agent with high affinity for prostate-specific membrane antigen. The coated fluorous silica kits are simple to prepare, and reactions can be performed at room temperature using different oxidants generating products in minutes in biocompatible solutions. PMID:25069901

  16. Purification of a native membrane-associated adenovirus tumor antigen.

    PubMed Central

    Persson, H; Katze, M G; Philipson, L

    1982-01-01

    A 15,000-dalton protein was purified from HeLa cells infected with adenovirus type 2. Proteins solubilized from a membrane fraction of lytically infected cells was used as the starting material for purification. Subsequent purification steps involved lentil-lectin, phosphocellulose, hydroxyapatite, DEAE-cellulose, and aminohexyl-Sepharose chromatographies. A monospecific antiserum, raised against the purified protein, immunoprecipitated a 15,000-dalton protein encoded in early-region E1B (E1B/15K protein) of the adenovirus type 2 DNA. Tryptic finger print analysis revealed that the purified protein was identical to the E1B/15K protein encoded in the transforming part of the viral genome. The antiserum immunoprecipitated the E1B/15K protein from a variety of viral transformed cell lines isolated from humans, rats, or hamsters. The E1B/15K protein was associated with the membrane fraction of both lytically and virus-transformed cell lines and could only be released by detergent treatment. Furthermore, a 11,000- to 12,000-dalton protein that could be precipitated with the anti-E1B/15K serum was recovered from membranes treated with trypsin or proteinase K, suggesting that a major part of the E1B/15K protein is protected in membrane vesicles. Translation of early viral mRNA in a cell-free system, supplemented with rough microsomes, showed that this protein was associated with the membrane fraction also in vitro. Images PMID:7097863

  17. The Twelve Steps Experientially.

    ERIC Educational Resources Information Center

    Horne, Lianne

    Experiential activities provide each participant with the ability to see, feel, and experience whatever therapeutic issue the facilitator is addressing, and usually much more. This paper presents experiential activities to address the 12 steps of recovery adopted from Alcoholics Anonymous. These 12 steps are used worldwide for many other recovery…

  18. Soluble expression, rapid purification, and characterization of human interleukin-24 (IL-24) using a MBP-SUMO dual fusion system in Escherichia coli.

    PubMed

    Zhang, Jian; Lv, Xinxin; Xu, Rui; Tao, Xinyi; Dong, Yuguo; Sun, Aiyou; Wei, Dongzhi

    2015-08-01

    Interleukin-24 (IL-24), a cytokine belonging to the IL-10 family, can selectively induce apoptosis in a broad range of tumor cells without harming normal cells. The efficient and soluble expression of bioactive recombinant IL-24 in Escherichia coli remains an obstacle because of aggregation and insufficient yield. In this study, a fusion of the small ubiquitin-related modifier (SUMO) or maltose-binding protein (MBP) has shown potential in facilitating the produce of IL-24. Thus, a new construct for MBP-SUMO-IL-24 expression would be a promising approach. Our results showed that the MBP-SUMO-IL-24 fusion protein was efficiently expressed as a soluble protein. SUMO protease-mediated cleavage at the SUMO/IL-24 junction released the recombinant IL-24 from the fusion protein. In addition, a His6 tag fused upstream of SUMO allowed for one-step purification through nickel affinity chromatography. Cleavage of the MBP-SUMO tag on the column resulted in the release of purified IL-24 and simplified the purification process. The final yield of IL-24 with approximately 90 % purity was 19 mg/L in flask fermentation. In vitro activity assays demonstrated that the purified IL-24 could induce apoptosis in MCF-7 breast cancer cells, but not normal NHLF cells, in a dose-dependent manner. In summary, we developed a novel method to express soluble and bioactive IL-24 protein in prokaryotic cells. PMID:25681151

  19. STEP Experiment Requirements

    NASA Technical Reports Server (NTRS)

    Brumfield, M. L. (Compiler)

    1984-01-01

    A plan to develop a space technology experiments platform (STEP) was examined. NASA Langley Research Center held a STEP Experiment Requirements Workshop on June 29 and 30 and July 1, 1983, at which experiment proposers were invited to present more detailed information on their experiment concept and requirements. A feasibility and preliminary definition study was conducted and the preliminary definition of STEP capabilities and experiment concepts and expected requirements for support services are presented. The preliminary definition of STEP capabilities based on detailed review of potential experiment requirements is investigated. Topics discussed include: Shuttle on-orbit dynamics; effects of the space environment on damping materials; erectable beam experiment; technology for development of very large solar array deployers; thermal energy management process experiment; photovoltaic concentrater pointing dynamics and plasma interactions; vibration isolation technology; flight tests of a synthetic aperture radar antenna with use of STEP.

  20. One-step detection of pathogens and cancer biomarkers by the naked eye based on aggregation of immunomagnetic beads

    NASA Astrophysics Data System (ADS)

    Chen, Yiping; Xianyu, Yunlei; Sun, Jiashu; Niu, Yajing; Wang, Yu; Jiang, Xingyu

    2015-12-01

    This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay.This report shows that immunomagnetic beads (IMBs) can act as the optical readout for assays, in addition to serving as the carrier for purification/separation. Under the influence of an external magnet, IMBs are attracted to coat one side of a test tube. IMBs specifically bound to targets can form a narrow brown stripe, whereas free IMBs will form a diffuse, yellow coating on the side of the test tube. Target analytes can aggregate initially dispersed IMBs in a sample concentration-dependent manner, yielding a color change from yellow to brown that can be seen with the naked eye. This assay combines the convenience of a lateral flow assay, allowing a one-step assay to finish within 15 min, with the sensitivity of an enzyme-linked immonosorbent assay. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07044a

  1. Updating of sewage - purification facilities of electroplating enterprises with counterflow ion-exchange filters

    NASA Astrophysics Data System (ADS)

    Torosyan, V. F.; Torosyan, E. S.; Sorokin, P. D.; Telitsyn, A. A.

    2015-09-01

    The paper focuses on work of electroplating sewage-purification facilities of mechanical engineering production; drawbacks caused by specific features of physical and chemical processes of coagulation and technological malfunctions have been revealed. Additional equipment - ion-exchanging filters have been selected on the basis of designed methods, they make it possible for enterprises of mechanical engineering to implement conversion to water rotation systems.

  2. Application of protein purification methods for the enrichment of a cytotoxin from Campylobacter jejuni

    PubMed Central

    2012-01-01

    report the enrichment and partial purification of C. jejuni cytotoxin by HPLC ion-exchange chromatography. Further purification may be achieved using additional complementary chromatographic techniques. A short-list of six candidate cytotoxin proteins was identified using an LCMS screen of pool B. Successful isolation of the cytotoxin will initiate steps for the determination of the role of this cytotoxin in the pathogenesis of C. jejuni diarrhoea. PMID:23259594

  3. Urate Oxidase Purification by Salting-in Crystallization: Towards an Alternative to Chromatography

    PubMed Central

    Giffard, Marion; Ferté, Natalie; Ragot, François; El Hajji, Mohamed; Castro, Bertrand; Bonneté, Françoise

    2011-01-01

    Background Rasburicase (Fasturtec® or Elitek®, Sanofi-Aventis), the recombinant form of urate oxidase from Aspergillus flavus, is a therapeutic enzyme used to prevent or decrease the high levels of uric acid in blood that can occur as a result of chemotherapy. It is produced by Sanofi-Aventis and currently purified via several standard steps of chromatography. This work explores the feasibility of replacing one or more chromatography steps in the downstream process by a crystallization step. It compares the efficacy of two crystallization techniques that have proven successful on pure urate oxidase, testing them on impure urate oxidase solutions. Methodology/Principal Findings Here we investigate the possibility of purifying urate oxidase directly by crystallization from the fermentation broth. Based on attractive interaction potentials which are known to drive urate oxidase crystallization, two crystallization routes are compared: a) by increased polymer concentration, which induces a depletion attraction and b) by decreased salt concentration, which induces attractive interactions via a salting-in effect. We observe that adding polymer, a very efficient way to crystallize pure urate oxidase through the depletion effect, is not an efficient way to grow crystals from impure solution. On the other hand, we show that dialysis, which decreases salt concentration through its strong salting-in effect, makes purification of urate oxidase from the fermentation broth possible. Conclusions The aim of this study is to compare purification efficacy of two crystallization methods. Our findings show that crystallization of urate oxidase from the fermentation broth provides purity comparable to what can be achieved with one chromatography step. This suggests that, in the case of urate oxidase, crystallization could be implemented not only for polishing or concentration during the last steps of purification, but also as an initial capture step, with minimal changes to the

  4. BODIPY–BODIPY dyad: assessing the potential as a viscometer for molecular and ionic liquids† †Electronic supplementary information (ESI) available: Details on the synthesis and purification of BODIPY dyes and ILs, sample preparations and physical properties of ILs as well as spectral data. See DOI: 10.1039/c4ra09757b Click here for additional data file.

    PubMed Central

    Kimball, Joseph D.; Raut, Sangram; Jameson, Laramie P.; Smith, Nicholas W.

    2015-01-01

    A symmetrical BODIPY–BODIPY dyad with a diyne linker was prepared in two steps; the lifetime decay of this rotor appeared to correlate with the viscosity of the media, thus making this dyad a suitable small molecule viscometer for molecular solvents. The potential of using the rotor to probe the viscosity of ionic liquids was also investigated. PMID:25844163

  5. Preparative isolation and purification of ginsenosides Rf, Re, Rd and Rb1 from the roots of Panax ginseng with a salt/containing solvent system and flow step-gradient by high performance counter-current chromatography coupled with an evaporative light scattering detector.

    PubMed

    Qi, Xiaocheng; Ignatova, Svetlana; Luo, Guoan; Liang, Qionglin; Jun, Frank Wu; Wang, Yiming; Sutherland, Ian

    2010-03-26

    Ginseng is a popular herb worldwide and has had varied uses in traditional Asian medicine for thousands of years. There are several different species of the herb, but all share the same constituents. Ginsenosides, the most extensively studied chemical components of ginseng, are generally considered to be one of the most important active ingredients of the plant. In this study, we have developed fast and efficient methodology for isolation of four known ginsenosides Rf, Rd, Re and Rb1 from Ginseng by high performance counter-current chromatography (HPCCC) coupled with evaporative light scattering detection (ELSD). The crude sample for HPCCC was purified firstly from a ginseng extraction using macroporous resin. The enriched saponin fraction (480 mg) was separated by using methylene chloride-methanol-5 mM aqueous ammonium acetate-isopropanol (6:2:4:3, v/v,) as the two-phase solvent system and yielded 10.7 mg of Rf, 11.0 mg of Rd, 13.4 mg of Re and 13.9 mg of Rb1. The purity of these ginsenosides was 99.2%, 88.3%, 93.7% and 91.8%, respectively assessed by HPLC-DAD-ELSD, and their structures were characterized by electrospray ionization mass spectrometry (ESI-MS) and compared with standards. Ammonium acetate was used to shorten the separation time and eliminate emulsification together with a flow step-gradient. The salt can be removed by re-dissolving the sample using acetone. PMID:20171644

  6. Isolation, purification and antioxidant activities of polysaccharides from Grifola frondosa.

    PubMed

    Chen, Gui-tang; Ma, Xue-mei; Liu, Sheng-to; Liao, Yan-li; Zhao, Guo-qang

    2012-06-01

    The crude polysaccharides (GFP) were isolated from the fruiting bodies of Grifola frondosa and purified by DEAE cellulose-52 chromatography and Sephadex G-100 size-exclusion chromatography in that order. Three main fractions, GFP-1, GFP-2 and GFP-3, were obtained through the isolation and purification steps. Then the antioxidant activities of these three fractions were investigated in vitro. The results showed that GFP-1, GFP-2 and GFP-3 possessed significant inhibitory effects on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical and superoxide radical; their reducing power, ferrous ions chelating effect and the inhibition ability of the rat liver lipid oxidation where also strong. These results suggest that G. frondosa polysaccharides could be a suitable natural antioxidant and may be the functional foods for humans. PMID:24750604

  7. Modified purification of mercuric iodide for crystal growth

    NASA Astrophysics Data System (ADS)

    Skinner, N. L.; Ortale, C.; Schieber, M. M.; Vandenberg, L.

    The standard procedure used in our laboratory to purify commercially available mercuric iodide consists of a sequence of steps: (1) repeated sublimation under continous evacuation, followed by (2) melting and recrystallization, and (3) a sublimation process in a closed tube. This paper describes a modification of the standard purification sequence by adding recrystallization of the mercuric iodide in hydrochloric acid. Leaching cation impurities out of mercuric iodide powder with hydrochloric acid has been practiced before by Zaletin, (V.M. Zaletin, I.H. Nozhiua, I.N. Fomin, V.T. Shystov, and N.V. Protasov, Atomic Energy 48, 169 (1980)). Our objective for the hydrochloric acid treatment was to remove nitrates and hydrocarbons which were interfering with the vapor transport during crystal growth. Results of the procedure are presented in terms of total carbon and selected ion content of the treated and untreated material.

  8. Modified purification of mercuric iodide for crystal growth

    NASA Astrophysics Data System (ADS)

    Skinner, N. L.; Ortale, C.; Schieber, M. M.; Van Den Berg, L.

    1988-06-01

    The standard procedure used in our laboratory to purify commercially available mercuric iodide (HgI 2) consists of a sequence of steps: (1) repeated sublimation under continuous evacuation, followed by (2) melting and recrystallization, and (3) a sublimation process in a closed tube. This paper describes a modification of the standard purification sequence by adding recrystallization of the HgI 2 in hydrochloric acid. Leaching cation impurities out of HgI 2 powder with hydrochloric acid has been practised before by Zaletin et al. Our objective for the hydrochloric acid treatment was to remove nitrates and hydrocarbons which were interfering with the vapor transport during crystal growth. Results of the procedure are presented in terms of total carbon and selected ion content of the treated and untreated material.

  9. Purification and characterization of an alkaline protease from Acetes chinensis

    NASA Astrophysics Data System (ADS)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15 folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55°C and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+, EDTA and PMSF could inhibit its activity.

  10. Magnetite decorated activated carbon composites for water purification

    NASA Astrophysics Data System (ADS)

    Barala, Sunil Kumar; Arora, Manju; Saini, Parveen

    2013-06-01

    Activated carbon decorated with magnetite (ACMG) nanoparticles composites have been prepared by facile method via impregnation of AC with stable dispersion of superparamagnetic MG nanoparticles followed by drying. These composites exhibit both magnetic and porosity behavior which can be easily optimized by controlling the weight ratio of two phases. The structural, magnetic, thermal and morphological properties of these as synthesized ACMG samples were characterized by powder XRD, FTIR, VSM and SEM techniques. The ACMG powder has been used for water purification having methylene blue (MB) dye as an impurity. The nanoporosity of these composites allow rapid adsorption of MB and their magnetic behavior helps in single step separation of MB adsorbed ACMG particles by the application of external magnetic field.

  11. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae

    PubMed Central

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  12. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae.

    PubMed

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  13. Integrated analysis and design optimization of germanium purification process using zone-refining technique

    NASA Astrophysics Data System (ADS)

    Wang, Sen; Fang, H. S.; Jin, Z. L.; Zhao, C. J.; Zheng, L. L.

    2014-12-01

    Germanium (Ge) is a preferred material in the fabrication of high-performance gamma radiation detector for spectroscopy in nuclear physics. To maintain an intrinsic region in which electrons and holes reach the contacts to produce a spectroscopic signal, germanium crystals are usually doped with lithium (Li) ions. Consequently, hyperpure germanium (HPGe) should be prepared before the doping process to eliminate the interference of unexpected impurities in the Li dopant. Zone-refining technique, widely used in purification of ultra-pure materials, is chosen as one of the purification steps during detector-grade germanium production. In the paper, numerical analysis has been conducted to analyze heat transfer, melt flow and impurity segregation during a multi-pass zone-refining process of germanium in a Cyberstar mirror furnace. By modifying the effective redistribution coefficients, axial segregations of various impurities are investigated. Marangoni convection is found dominant in the melt. It affects the purification process through modifying the boundary layer thickness. Impurity distributions along the ingot are obtained with different conditions, such as pass number, zone travel rate, initial impurity concentration, segregation coefficient, and hot-zone length. Based on the analysis, optimization of the purification process design is proposed.

  14. Combustion water purification techniques influence on OBT analysing using liquid scintillation counting method

    SciTech Connect

    Varlam, C.; Vagner, I.; Faurescu, I.; Faurescu, D.

    2015-03-15

    In order to determine organically bound tritium (OBT) from environmental samples, these must be converted into water, measurable by liquid scintillation counting (LSC). For this purpose we conducted some experiments to determine OBT level of a grass sample collected from an uncontaminated area. The studied grass sample was combusted in a Parr bomb. However usual interfering phenomena were identified: color or chemical quench, chemiluminescence, overlap over tritium spectrum because of other radionuclides presence as impurities ({sup 14}C from organically compounds, {sup 36}Cl as chloride and free chlorine, {sup 40}K as potassium cations) and emulsion separation. So the purification of the combustion water before scintillation counting appeared to be essential. 5 purification methods were tested: distillation with chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}), lyophilization, chemical treatment (Na{sub 2}O{sub 2} and KMnO{sub 4}) followed by lyophilization, azeotropic distillation with toluene and treatment with a volcanic tuff followed by lyophilization. After the purification step each sample was measured and the OBT measured concentration, together with physico-chemical analysis of the water analyzed, revealed that the most efficient method applied for purification of the combustion water was the method using chemical treatment followed by lyophilization.

  15. Affinity purification of metalloprotease from marine bacterium using immobilized metal affinity chromatography.

    PubMed

    Li, Shangyong; Wang, Linna; Yang, Juan; Bao, Jing; Liu, Junzhong; Lin, Shengxiang; Hao, Jianhua; Sun, Mi

    2016-06-01

    In this study, an efficient affinity purification protocol for an alkaline metalloprotease from marine bacterium was developed using immobilized metal affinity chromatography. After screening and optimization of the affinity ligands and spacer arm lengths, Cu-iminmodiacetic acid was chosen as the optimal affinity ligand, which was coupled to Sepharose 6B via a 14-atom spacer arm. The absorption analysis of this medium revealed a desorption constant Kd of 21.5 μg/mL and a theoretical maximum absorption Qmax of 24.9 mg/g. Thanks to this affinity medium, the enzyme could be purified by only one affinity purification step with a purity of approximately 95% pure when analyzed by high-performance liquid chromatography and reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The recovery of the protease activity reached 74.6%, which is much higher than the value obtained by traditional protocols (8.9%). These results contribute to the industrial purifications and contribute a significant reference for the purification of other metalloproteases. PMID:27058973

  16. 2-Step IMAT and 2-Step IMRT in three dimensions

    SciTech Connect

    Bratengeier, Klaus

    2005-12-15

    In two dimensions, 2-Step Intensity Modulated Arc Therapy (2-Step IMAT) and 2-Step Intensity Modulated Radiation Therapy (IMRT) were shown to be powerful methods for the optimization of plans with organs at risk (OAR) (partially) surrounded by a target volume (PTV). In three dimensions, some additional boundary conditions have to be considered to establish 2-Step IMAT as an optimization method. A further aim was to create rules for ad hoc adaptations of an IMRT plan to a daily changing PTV-OAR constellation. As a test model, a cylindrically symmetric PTV-OAR combination was used. The centrally placed OAR can adapt arbitrary diameters with different gap widths toward the PTV. Along the rotation axis the OAR diameter can vary, the OAR can even vanish at some axis positions, leaving a circular PTV. The width and weight of the second segment were the free parameters to optimize. The objective function f to minimize was the root of the integral of the squared difference of the dose in the target volume and a reference dose. For the problem, two local minima exist. Therefore, as a secondary criteria, the magnitude of hot and cold spots were taken into account. As a result, the solution with a larger segment width was recommended. From plane to plane for varying radii of PTV and OAR and for different gaps between them, different sets of weights and widths were optimal. Because only one weight for one segment shall be used for all planes (respectively leaf pairs), a strategy for complex three-dimensional (3-D) cases was established to choose a global weight. In a second step, a suitable segment width was chosen, minimizing f for this global weight. The concept was demonstrated in a planning study for a cylindrically symmetric example with a large range of different radii of an OAR along the patient axis. The method is discussed for some classes of tumor/organ at risk combinations. Noncylindrically symmetric cases were treated exemplarily. The product of width and weight of

  17. Monoclonal antibody purification with hydroxyapatite.

    PubMed

    Gagnon, Pete

    2009-06-01

    Hydroxyapatite (HA) has been used for IgG purification since its introduction in the 1950s. Applications expanded to include IgA and IgM in the 1980s, along with elucidation of its primary binding mechanisms and the development of ceramic HA media. With the advent of recombinant monoclonal antibodies, HA was demonstrated to be effective for removal of antibody aggregates, as well as host cell proteins and leached protein A. HA's inherent abilities have been enhanced by the development of elution strategies that permit differential control of its primary binding mechanisms: calcium metal affinity and phosphoryl cation exchange. These strategies support reduction of antibody aggregate content from greater than 60% to less than 0.1%, in conjunction with enhanced removal of DNA, endotoxin, and virus. HA also has a history of discriminating various immunological constructs on the basis of differences in their variable regions, or discriminating Fab fragments from Fc contaminants in papain digests of purified monoclonal IgG. Continuing development of novel elution strategies, alternative forms of HA, and application of robotic high throughput screening systems promise to expand HA's utility in the field. PMID:19491046

  18. Dialysis membranes for blood purification.

    PubMed

    Sakai, K

    2000-01-01

    All of the artificial membranes in industrial use, such as a reverse-osmosis membrane, dialysis membrane, ultrafiltration membrane, microfiltration membrane and gas separation membrane, also have therapeutic applications. The most commonly used artificial organ is the artificial kidney, a machine that performs treatment known as hemodialysis. This process cleanses the body of a patient with renal failure by dialysis and filtration, simple physicochemical processes. Hemodialysis membranes are used to remove accumulated uremic toxins, excess ions and water from the patient via the dialysate, and to supply (deficit) insufficient ions from the dialysate. Dialysis membranes used clinically in the treatment of patients with renal failure account for by far the largest volume of membranes used worldwide; more than 70 million square meters are used a year. Almost all dialyzers now in use are of the hollow-fiber type. A hollow-fiber dialyzer contains a bundle of approximately 10000 hollow fibers, each with an inner diameter of about 200 microm when wet. The membrane thickness is about 20-45 microm, and the length is 160-250 mm. The walls of the hollow fibers function as the dialysis membrane. Various materials, including cellulose-based materials and synthetic polymers, are used for dialysis membranes. This paper reviews blood purification, hemodialysis and dialysis membranes. PMID:10898241

  19. Purification, amino acid sequence and mode of action of bifidocin B produced by Bifidobacterium bifidum NCFB 1454.

    PubMed

    Yildirim, Z; Winters, D K; Johnson, M G

    1999-01-01

    Bifidocin B produced by Bifidobacterium bifidum NCFB 1454 was purified to homogeneity by a rapid and simple three step purification procedure which included freeze drying, Micro-Cel adsorption/desorption and cation exchange chromatography. The purification resulted in 18% recovery and an approximately 1900-fold increase in the specific activity and purity of bifidocin B. Treatment with bifidocin B caused sensitive cells to lose high amounts of intracellular K+ ions and u.v.-absorbing materials, and to become more permeable to ONPG. Bifidocin B adsorbed to the Gram-positive bacteria but not the Gram-negative bacteria tested. Its adsorption was pH-dependent but not time-dependent. For sensitive cells, the adsorption and lethal action of bifidocin B was very rapid. In 5 min, 95% of bifidocin B adsorbed onto sensitive cells. Several salts inhibited the binding of bifidocin B, which could be overcome by increasing the amount of bifidocin B added. Pre-treatment of sensitive cells and cell walls with detergents, organic solvents or enzymes did not cause a reduction in subsequent cellular binding of bifidocin B, but cell wall preparations treated with methanol:chloroform and hot 20% (w/v) TCA lost the ability to adsorb bifidocin B. Also, the addition of purified heterologous lipoteichoic acid to sensitive cells completely blocked the adsorption of bifidocin B. The amino acid sequence indicated that the bacteriocin contained 36 residues. N-terminal amino acid sequence analysis yielded a sequence of KYYGNGVTCGLHDCRVDRGKATCGIINNGGMWGDIG. Curing experiments with 20 micrograms ml-1 acriflavine yielded cell derivatives that no longer produced bifidocin B but retained immunity to bifidocin B. Production of bifidocin B, but not immunity to bifidocin B, was associated with a plasmid of about 8 kb in this strain. PMID:10030011

  20. The Next Giant Step

    NASA Video Gallery

    Artist Robert McCall painted "The Next Giant Step" in 1979 to commemorate the heroism and courage of spaceflight pioneers. Located in the lobby of Johnson's building 2, the mural depicts America's ...

  1. Purification of silicon for photovoltaic applications

    NASA Astrophysics Data System (ADS)

    Delannoy, Yves

    2012-12-01

    Solar grade silicon, as a starting material for crystallization to produce solar cells, is discussed here in terms of impurities whose maximum content is estimated from recent literature and conferences. A review of the production routes for each category of solar-grade silicon (undoped, compensated or heavily compensated) is proposed with emphasis on the metallurgical route. Some recent results are proposed concerning segregation, showing that directional solidification systems can be used for solidification even at high solidification rate (15 cm/h). Results on inductive plasma purification, where boron is evacuated as HBO in a gas phase blown from an inductive plasma torch, are shown to apply as well to arc plasmas and purification by moist gas. Special attention is paid to the history of impurities in the purification processes, showing that impure auxiliary phases (silicon tetrachloride, slag, aluminum, etc.) often need their own purification process to enable their recycling, which has to be considered to evaluate the cost (financial, energetic and environmental) of the purification route.

  2. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is...

  3. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is...

  4. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is...

  5. 21 CFR 876.5665 - Water purification system for hemodialysis.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Water purification system for hemodialysis. 876.5665 Section 876.5665 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN... purification system for hemodialysis. (a) Identification. A water purification system for hemodialysis is...

  6. Carbohydrate-mediated purification of petrochemicals.

    PubMed

    Holcroft, James M; Hartlieb, Karel J; Moghadam, Peyman Z; Bell, Jon G; Barin, Gokhan; Ferris, Daniel P; Bloch, Eric D; Algaradah, Mohammed M; Nassar, Majed S; Botros, Youssry Y; Thomas, K Mark; Long, Jeffrey R; Snurr, Randall Q; Stoddart, J Fraser

    2015-05-01

    Metal-organic frameworks (MOFs) are known to facilitate energy-efficient separations of important industrial chemical feedstocks. Here, we report how a class of green MOFs-namely CD-MOFs-exhibits high shape selectivity toward aromatic hydrocarbons. CD-MOFs, which consist of an extended porous network of γ-cyclodextrins (γ-CDs) and alkali metal cations, can separate a wide range of benzenoid compounds as a result of their relative orientation and packing within the transverse channels formed from linking (γ-CD)6 body-centered cuboids in three dimensions. Adsorption isotherms and liquid-phase chromatographic measurements indicate a retention order of ortho- > meta- > para-xylene. The persistence of this regioselectivity is also observed during the liquid-phase chromatography of the ethyltoluene and cymene regioisomers. In addition, molecular shape-sorting within CD-MOFs facilitates the separation of the industrially relevant BTEX (benzene, toluene, ethylbenzene, and xylene isomers) mixture. The high resolution and large separation factors exhibited by CD-MOFs for benzene and these alkylaromatics provide an efficient, reliable, and green alternative to current isolation protocols. Furthermore, the isolation of the regioisomers of (i) ethyltoluene and (ii) cymene, together with the purification of (iii) cumene from its major impurities (benzene, n-propylbenzene, and diisopropylbenzene) highlight the specificity of the shape selectivity exhibited by CD-MOFs. Grand canonical Monte Carlo simulations and single component static vapor adsorption isotherms and kinetics reveal the origin of the shape selectivity and provide insight into the capability of CD-MOFs to serve as versatile separation platforms derived from renewable sources. PMID:25806952

  7. Plasmon-enhanced photocatalytic water purification.

    PubMed

    Gomez, Leyre; Sebastian, Victor; Arruebo, Manuel; Santamaria, Jesus; Cronin, Stephen B

    2014-08-01

    Increasing water demand and water scarcity around the world requires the development of robust and efficient methods for water purification in the coming decades. Here, we report a photocatalytic water purification method using visible light (532 nm) utilizing 5 nm gold nanoparticles and their enhancement when attached on the surface of silica nanospheres as an inactive support to prevent nanoparticle coalescence or sintering. This is a non-toxic, low-cost, and easy photocatalytic process which provides high decomposition rates. Decomposition of the methyl orange dye is tested as a reaction model and trichloroethylene is selected as an example of a real water pollutant. When irradiated at their plasmon resonant frequency, the gold nanoparticles generate hydroxyl radicals that degradate organic pollutants into non-toxic molecules representing a basic mechanism of photocatalytic water purification. PMID:24590124

  8. Purification of specific loci for proteomic analysis

    PubMed Central

    Byrum, Stephanie D.; Taverna, Sean D.; Tackett, Alan J.

    2015-01-01

    Purification of small, native chromatin sections for proteomic identification of specifically bound proteins and histone posttranslational modifications is a powerful approach for studying mechanisms of chromosome metabolism. We detail a Chromatin Affinity Purification with Mass Spectrometry (ChAP-MS) approach for affinity purification of ~1 kb sections of chromatin for targeted proteomic analysis. This approach utilizes quantitative, high resolution mass spectrometry to categorize proteins and histone posttranslational modifications co-enriching with the given chromatin section as either “specific” to the targeted chromatin or “non-specific” contamination. In this way, the ChAP-MS approach can help define and re-define mechanisms of chromatin-templated activities. PMID:25311124

  9. Purification of glycocalicin from human plasma.

    PubMed

    HadjKacem, Basma; Mkaouar, Héla; Ben Amor, Ikram; Gargouri, Jalel; Gargouri, Ali

    2016-01-01

    Glycocalicin (GC) is a large extracellular proteolytic fragment of glycoprotein Ib, a membrane platelet component playing an essential role in the physiological processes of platelet adhesion and aggregation. GC contains the binding sites for thrombin and von Willebrand factor. GC circulates normally in vivo in significant concentrations and the plasma level of this protein reflects a complex function of factors including platelet count or platelet turnover. It can therefore serve as a good indicator for many diseases like hypoplastic thrombocytopenia and idiopathic thrombocytopenic purpura. For this reason, several purification assays have been previously described. In this work, we describe a novel analytical method for GC purification from human platelets based on preparative HPLC gel filtration followed by immuno-affinity chromatography on NHS activated column conjugated with specific antibody. Pure GC was obtained from tiny amount of starting material. Our protocol of GC purification is simple, fast and provides a pure end product. PMID:26606109

  10. Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry.

    PubMed

    Zhang, Qingchun; Goetze, Andrew M; Cui, Huanchun; Wylie, Jenna; Trimble, Steve; Hewig, Art; Flynn, Gregory C

    2014-01-01

    An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms. PMID:24518299

  11. Purification of the c-erbB2/neu membrane-spanning segment: a hydrophobic challenge.

    PubMed

    Goetz, M; Rusconi, F; Belghazi, M; Schmitter, J M; Dufourc, E J

    2000-01-14

    High quality purification of membrane-spanning peptides and proteins remains a challenging problem. In this work we describe a tailored chromatographic purification of a synthetic 35-residue peptide corresponding to the transmembrane region of the tyrosine kinase receptor c-erb2/neu. Composed to over 70% by the amino acids alanine, isoleucine, leucine, phenylalanine and valine, this peptide presents a very hydrophobic character. Product isolation from the complex peptide mixture, obtained after acid cleavage of the resin matrix used during the solid-phase synthesis, represents a difficult task. We propose a three step strategy based on gel permeation and reversed-phase high-performance liquid chromatography, using aprotic polar solvent mixtures. The challenge consisted in obtaining a sufficient amount of an extremely pure sample, in order to allow structural analysis by NMR spectroscopy. Keeping trace of the synthetic peptide throughout the different purification steps was assured by MALDI TOF mass spectrometry, and the final product purity was checked by coupled liquid chromatography-ESI TOF mass spectrometry. PMID:10681041

  12. Purification of Crystallization-Grade RNA Polymerase I from S. cerevisiae.

    PubMed

    Engel, Christoph

    2016-01-01

    Purification of RNA polymerase (Pol) I is essential for functional as well as for structural studies. The product needs to be extremely pure in order to exclude secondary effects, e.g., caused by copurified nucleic acids in subsequent experiments. For this purpose, the method presented here was originally introduced nearly a decade ago but underwent constant optimization [1]. The polymerase is extracted from its endogenous source, since no overexpression system for the entire 590 kDa, 14-subunit complex is available thus far. Following yeast cultivation, a number of standard protein purification techniques are applied and combined to a robust but elaborate procedure that takes 3 days. In brief, a yeast strain with histidine-tagged RNA polymerase I is fermented, cells are broken by bead beating, and cell debris is removed by a two-step centrifugation. The lysate is then dialyzed, the Pol-I-containing pellet resuspended, and polymerase I enriched by a His-trap affinity step, followed by sequential purification via anion and cation exchange and a final size exclusion chromatography. PMID:27576712

  13. Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

    PubMed Central

    Zhang, Qingchun; Goetze, Andrew M; Cui, Huanchun; Wylie, Jenna; Trimble, Steve; Hewig, Art; Flynn, Gregory C

    2014-01-01

    An advanced two-dimensional liquid chromatography/mass spectrometry platform was used to quantify individual host cell proteins (HCPs) present at various purification steps for several therapeutic monoclonal antibodies (mAbs) produced in Chinese hamster ovary cells. The methodology produced reproducible identifications and quantifications among replicate analyses consistent with a previously documented individual limit of quantification of ~13 ppm. We were able to track individual HCPs from cell culture fluid to protein A eluate pool to subsequent viral inactivation pool and, in some cases, further downstream. Approximately 500 HCPs were confidently identified in cell culture fluid and this number declined progressively through the purification scheme until no HCPs could be confidently identified in polishing step cation-exchange eluate pools. The protein A eluate pool of nine different mAbs contained widely differing numbers, and total levels, of HCPs, yet the bulk of the total HCP content in each case consisted of a small subset of normally intracellular HCPs highly abundant in cell culture fluid. These observations hint that minimizing cell lysis during cell culture/harvest may be useful in minimizing downstream HCP content. Clusterin and actin are abundant in the protein A eluate pools of most mAbs studied. HCP profiling by this methodology can provide useful information to process developers and lead to the refinement of existing purification platforms. PMID:24518299

  14. Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli.

    PubMed

    Wingfield, Paul T; Palmer, Ira; Liang, Shu-Mei

    2014-01-01

    Heterologous expression of recombinant proteins in E. coli often results in the formation of insoluble and inactive protein aggregates, commonly referred to as inclusion bodies. To obtain the native (i.e., correctly folded) and hence active form of the protein from such aggregates, four steps are usually followed: (1) the cells are lysed, (2) the cell wall and outer membrane components are removed, (3) the aggregates are solubilized (or extracted) with strong protein denaturants, and (4) the solubilized, denatured proteins are folded with concomitant oxidation of reduced cysteine residues into the correct disulfide bonds to obtain the native protein. This unit features three different approaches to the final step of protein folding and purification. In the first, guanidine·HCl is used as the denaturant, after which the solubilized protein is folded (before purification) in an "oxido-shuffling" buffer system to increase the rate of protein oxidation. In the second, acetic acid is used to solubilize the protein, which is then partially purified by gel filtration before folding; the protein is then folded and oxidized by simple dialysis against water. Thirdly, folding and purification of a fusion protein using metal-chelate affinity chromatography are described. PMID:25367010

  15. Partial purification and properties of cyclodextrin glycosiltransferase (CGTase) from alkalophilic Bacillus species.

    PubMed

    Mora, Marlene M Martínez; Sánchez, Karel Hernández; Santana, Reynaldo Villalonga; Rojas, Arley Pérez; Ramírez, Héctor L; Torres-Labandeira, Juan José

    2012-12-01

    Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.9) is an unique enzyme capable of converting starch and related substrates into cyclodextrins (CDs). In this paper, we report an one step gel purification method of CGTase from Bacillus sp. and later enzyme characterization. The Bacillus sp. strain was isolated from a Colocacia esculenta rizospheric soil sample and the CGTase production was carried out in alkaline medium (pH=10). The CGTase purification from the culture supernatant was performed by gel filtration. The enzyme was purified in one step with a recovery of 87.3% activity and 40-fold purification for specific enzymatic activity of 2.24 U/mg. Optimal activity was observed at pH 5.0 in citrate-phosphate buffer, and the enzyme retained almost 100 % of its activity between pH 5.5 and 10 after incubation for 1 h at 4°C. The enzyme exhibited maximum activity at 55°C and showed a T(50%) of 70°C. The ratio of α:β:γ CD formed by the enzyme was 0.74:1:0.61 for soluble starch and 0.29:1:0.85 for cocoyam starch. PMID:23420668

  16. Thermochemical Analysis for Purification of Polysilicon Melts

    SciTech Connect

    Ho, Pauline: Gee, James M.

    1999-05-01

    Chemical Equilibrium calculations are presented that are relevant to the purification of molten silicon by gas-blowing. The equilibrium distributions of silicon, boron, phosphorus carbon and iron among the solid, liquid and gas phases are reported for a variety of added chemicals, temperatures and total pressures. The identities of the dominant chemical species for each element in each phase are also provided for these conditions. The added gases examined are O(2), air, water, wet air, HCl, Cl(2), Cl(2)/O(2), SiCl(4), NH(3), NH(4)OH, and NH(4)Cl. These calculations suggest possible purification schemes, although kinetic or transport limitations may prove to be significant

  17. Isolation and purification of two antioxidant isomers of resveratrol dimer from the wine grape by counter-current chromatography.

    PubMed

    Kong, Qingjun; Ren, Xueyan; Hu, Ruilin; Yin, Xuefeng; Jiang, Guoshan; Pan, Yuanjiang

    2016-06-01

    Resveratrol dimers belong to a group of compounds called stilbenes, which along with proanthocyanidins, anthocyanins, catechins, and flavonols are natural phenolic compounds found in grapes and red wine. Stilbenes have a variety of structural isomers, all of which exhibit various biological properties. Counter-current chromatography with a two-phase solvent system composed of n-hexane/ethyl acetate/methanol/water (2:5:4:5, v/v/v/v) was applied to isolate and purify stilbene from the stems of wine grape. Two isomers of resveratrol dimers trans-ε-viniferin and trans-δ-viniferin were obtained from the crude sample in a one-step separation, with purities of 93.2 and 97.5%, respectively, as determined by high-performance liquid chromatography. The structures of these two compounds were identified by (1) H and (13) C NMR spectroscopy. In addition, their antioxidant activities were assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The antioxidant activities of trans-δ-viniferin were higher than that of trans-ε-viniferin in this model. This work demonstrated that counter-current chromatography is a powerful and effective method for the isolation and purification of polyphenols from wine grape. Additionally, the DPPH radical assay showed that the isolated component trans-δ-viniferin exhibited stronger antioxidant activities than trans-ε-viniferin and a little bit weaker than vitamin E at the same concentration. PMID:27130423

  18. Purification of F plasmid-encoded native TraC from Escherichia coli by affinity chromatography on calmodulin Sepharose.

    PubMed

    Hellstern, Simon; Mutzel, Rupert

    2016-06-01

    We have enriched several native bacterial proteins from Escherichia coli by chromatography on the immobilized eukaryotic Ca(2+)-binding protein, calmodulin. These bacterial proteins bound in a Ca(2+)-dependent manner to calmodulin, and were released by the addition of the Ca(2+)-chelator, EGTA, similar to many eukaryotic calmodulin-binding proteins. One of the bacterial proteins, F factor-encoded TraC, was purified to apparent homogeneity by an additional chromatographic step, anion exchange chromatography on MonoQ. Experiments with four chemically distinct calmodulin antagonists (R24571, Compound 48/80, melittin, and W7) showed that all of these substances inhibited the binding of purified TraC to calmodulin at effective concentrations comparable to those required for inhibiting in vitro binding of eukaryotic calmodulin-binding proteins. Three further bacterial proteins were identified as calmodulin-binding proteins: SecA, GlpD, and GlpC. We suggest that also these native bacterial proteins might be isolated by the unusual purification procedure including affinity chromatography on calmodulin Sepharose. Whether the identified proteins bind to, and are regulated by, putative bacterial calmodulin-like proteins in Escherichia coli remains to be established. PMID:26892535

  19. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity.

    PubMed

    Lane, Michael D; Seelig, Burckhard

    2016-05-01

    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures. PMID:26773742

  20. Purification of phage display-modified bacteriophage T4 by affinity chromatography

    PubMed Central

    2011-01-01

    Background Affinity chromatography is one of the most efficient protein purification strategies. This technique comprises a one-step procedure with a purification level in the order of several thousand-fold, adaptable for various proteins, differentiated in their size, shape, charge, and other properties. The aim of this work was to verify the possibility of applying affinity chromatography in bacteriophage purification, with the perspective of therapeutic purposes. T4 is a large, icosahedral phage that may serve as an efficient display platform for foreign peptides or proteins. Here we propose a new method of T4 phage purification by affinity chromatography after its modification with affinity tags (GST and Histag) by in vivo phage display. As any permanent introduction of extraneous DNA into a phage genome is strongly unfavourable for medical purposes, integration of foreign motifs with the phage genome was not applied. The phage was propagated in bacteria expressing fusions of the phage protein Hoc with affinity tags from bacterial plasmids, independently from the phage expression system. Results Elution profiles of phages modified with the specific affinity motifs (compared to non-specific phages) document their binding to the affinity resins and effective elution with standard competitive agents. Non-specific binding was also observed, but was 102-105 times weaker than the specific one. GST-modified bacteriophages were also effectively released from glutathione Sepharose by proteolytic cleavage. The possibility of proteolytic release was designed at the stage of expression vector construction. Decrease in LPS content in phage preparations was dependent on the washing intensity; intensive washing resulted in preparations of 11-40 EU/ml. Conclusions Affinity tags can be successfully incorporated into the T4 phage capsid by the in vivo phage display technique and they strongly elevate bacteriophage affinity to a specific resin. Affinity chromatography can be

  1. Novel thermo-responsive fucose binding ligands for glycoprotein purification by affinity precipitation.

    PubMed

    Arnold, Lindsay; Chen, Rachel

    2014-02-01

    Novel thermo-responsive affinity sugar binders were developed by fusing a bacterial fucose lectin with a thermo-responsive polypeptide. These designer affinity ligand fusions were produced using an Escherichia coli system capable of extracellular secretion of recombinant proteins and were isolated with a high recovery yield (95%) directly from growth medium by Inverse Temperature Cycling (ITC). With horse radish peroxidase (HRP) as a model protein, we demonstrate here that the designer thermo-responsive ligands are capable of interacting with glycans on a glycoprotein, a property that was used to develop a novel affinity precipitation method for glycoprotein purification. The method, requiring only simple process steps, affords full recovery of a target glycoprotein, and is effective at a target glycoprotein concentration as low as 1.4 pM in the presence of large amounts of contaminants. By developing other sugar binders in the similar fashion, the method should be highly useful for glycoprotein purification and detection. PMID:25271333

  2. Deterministic Polarization Entanglement Purification of W State in Multiple Degrees of Freedom with Hyper-Entanglement

    NASA Astrophysics Data System (ADS)

    Kuang, Hongyan; Guo, Ying

    2015-01-01

    We present two deterministic entanglement purification protocols for distilling W state in polarization entanglement in a one-step fashion. The three parties can obtain a maximally entangled W polarization state with success probability 100 % from the hyper-entangled W state in multiple degrees of freedom (DOFs), and they do not require the quantum system to be entangled in polarization DOF but in spatial DOF. The former protocol is implemented with the passive linear optics, which is feasible with current experiments. The later one is implemented with cross-Kerr-nonlinearities, which can achieve higher efficiency of the entanglement transformation among different DOFs since it does not require the sophisticated single-photon detector. The present protocols are both suitable for polarization entanglement purification of multi-photon-entangled W state in quantum computation network because they need less operations and classical communications in the practical implementation.

  3. Overexpression, purification and characterization of a recombinant secretary catalase from Bacillus subtilis.

    PubMed

    Shi, Xunlong; Feng, Meiqing; Zhao, Yujie; Guo, Xin; Zhou, Pei

    2008-01-01

    A recombinant Bacillus subtilis strain (KN25) was generated for the large-scale preparation of catalase. The B. subtilis katA gene encoding for catalase was cloned into the shuttle vector PRB374, downstream of the constitutively active vegII promoter, followed by transformation of the B. subtilis strain WB600 with the plasmid. The transformant strain, KN25 secretes high levels (3,500 U/ml) of catalase, which facilitates its purification. Three simple purification steps yielded nearly homogeneous catalase, with approximately 70% recovery. The purified recombinant catalase has a specific activity of 34,600 U/mg under optimal conditions, and is more resistant to acidic conditions than bovine liver catalase. PMID:17876537

  4. Purification of polyphenol oxidase free of the storage protein patatin from potato tuber.

    PubMed

    Partington, J C; Bolwell, G P

    1996-08-01

    Routine protein purification to homogeneity from potato tuber, as from other storage tissues and seeds, is often hindered due to the large amounts of storage protein present. In potato, patatin, the major storage protein of the tuber, often contaminates preparations. The present work describes the purification of polyphenol oxidase (PPO) from the potato tuber (Solanum tuberosum cv Cara) to homogeneity including the critical step of hydrophobic chromatography on Octyl-Sepharose which was sufficient to completely remove patatin. The purified PPO was found to be a doublet of M(r) 60,000 and 69,000 when analysed by SDS-PAGE with a Km 4.3 +/- 0.3 mM for L-dihydroxyphenylalanine. Both bands were found to have similar N-terminal corresponding to PPO isoforms when sequenced. PMID:8783836

  5. Differential fusion expression and purification of a cystatin in two different bacterial strains.

    PubMed

    Gholizadeh, A

    2013-01-01

    To date, the identification of the novel multifunctional properties of cysteine proteinase inhibitors "known as cystatins" is the great of interests for molecular biologists. The efficient production, purification and correctly folded form of these proteins are the most important requirements for their any basic research. To the best of our knowledge, maltose-binding protein (MBP) fusion tags are being used to overcome the impediment to their heterologous recombinant expression in Escherichia coli as insoluble and bio-inactive inclusion bodies. In the present work, to evaluate the expression efficiency of a cystatin molecule in E. coli cells by using MBP tags, the expression of Celosia cystatin was studied in two different strains of this bacterium. The quantitative analysis results based on the one-step purification yield of the fused product showed the excellency of the E. coli TB1 strain in comparison to E. coli DH5alpha for the high-level production of active product. PMID:24455860

  6. Classification and purification of proteins of heterogeneous nuclear ribonucleoprotein particles by RNA-binding specificities

    SciTech Connect

    Swanson, M.S.; Dreyfuss, G.

    1988-05-01

    Several proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles display very high binding affinities for different ribonucleotide homopolymers. The specificity of some of these proteins at high salt concentrations and in the presence of heparin allows for their rapid one-step purification from HeLa nucleoplasm. The authors show that the hnRNP proteins are poly(U)-binding proteins and compare their specificity to that of the previously described cytoplasmic poly(A)-binding protein. These findings provide a useful tool for the classification and purification of hnRNP proteins from various tissues and organisms and indicate that different hnRNP proteins have different RNA-binding specificities.

  7. Purification and amino acid sequence of aminopeptidase P from pig kidney.

    PubMed

    Vergas Romero, C; Neudorfer, I; Mann, K; Schäfer, W

    1995-04-01

    Aminopeptidase P from kidney cortex was purified in high yield (recovery greater than or equal to 20%) by a series of column chromatographic steps after solubilization of the membrane-bound glycoprotein with n-butanol. A coupled enzymic assay, using Gly-Pro-Pro-NH-Nap as substrate and dipeptidyl-peptidase IV as auxilliary enzyme, was used to monitor the purification. The purification procedure yielded two forms of aminopeptidase P differing in their carbohydrate composition (glycoforms). Both enzyme preparations were homogeneous as assessed by SDS/PAGE silver staining, and isoelectric focusing. Both forms possessed the same substrate specificity, catalysed the same reaction, and consisted of identical protein chains. The amino acid sequence determined by Edman degradation and mass spectrometry consisted of 623 amino acids. Six N-glycosylation sites, all contained in the N-terminal half of the protein, were characterized. PMID:7744038

  8. Automated centrifugal-microfluidic platform for DNA purification using laser burst valve and coriolis effect.

    PubMed

    Choi, Min-Seong; Yoo, Jae-Chern

    2015-04-01

    We report a fully automated DNA purification platform with a micropored membrane in the channel utilizing centrifugal microfluidics on a lab-on-a-disc (LOD). The microfluidic flow in the LOD, into which the reagents are injected for DNA purification, is controlled by a single motor and laser burst valve. The sample and reagents pass successively through the micropored membrane in the channel when each laser burst valve is opened. The Coriolis effect is used by rotating the LOD bi-directionally to increase the purity of the DNA, thereby preventing the mixing of the waste and elution solutions. The total process from the lysed sample injection into the LOD to obtaining the purified DNA was finished within 7 min with only one manual step. The experimental result for Salmonella shows that the proposed microfluidic platform is comparable to the existing devices in terms of the purity and yield of DNA. PMID:25737025

  9. Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose.

    PubMed

    DiScipio, Richard G; Liddington, Robert C; Schraufstatter, Ingrid U

    2016-05-01

    A method is reported to purify Fibulin-1 from human plasma resulting in a 36% recovery. The steps involve removal of the cryoglobulin and the vitamin K dependent proteins followed by polyethylene glycol and ammonium sulfate precipitations, DEAE-Sephadex column chromatography and finally Factor H-Sepharose affinity purification. The procedure is designed to be integrated into an overall scheme for the isolation of over 30 plasma proteins from a single batch of human plasma. Results from mass spectroscopy, SDS-PAGE, and Western blotting indicate that human plasma Fibulin-1 is a single chain of the largest isotype. Functional binding assays demonstrated calcium ion dependent interaction of Fibulin-1 for fibrinogen, fibronectin, and Factor H. The procedure described is the first to our knowledge that enables a large scale purification of Fibulin-1 from human plasma. PMID:26826315

  10. Stepped inlet optical panel

    DOEpatents

    Veligdan, James T.

    2001-01-01

    An optical panel includes stacked optical waveguides having stepped inlet facets collectively defining an inlet face for receiving image light, and having beveled outlet faces collectively defining a display screen for displaying the image light channeled through the waveguides by internal reflection.

  11. Steps Toward Effective Assessment.

    ERIC Educational Resources Information Center

    Cope, Carolyn O.

    1996-01-01

    Describes and defines the steps involved in measurement and evaluation: (1) determining an outcome; (2)defining scoring criteria; (3)establishing appropriate assessment tasks; and (4)creating opportunities for learning. Includes a flow chart for a design-down curriculum and an example of a vocal performance rating scale assessment. (MJP)

  12. Recovery and purification of chitosanase produced by Bacillus cereus using expanded bed adsorption and central composite design.

    PubMed

    de Araújo, Nathália Kelly; Pimentel, Vanessa Carvalho; da Silva, Nayane Macedo Portela; de Araújo Padilha, Carlos Eduardo; de Macedo, Gorete Ribeiro; Dos Santos, Everaldo Silvino

    2016-02-01

    This study presents a system for expanded bed adsorption for the purification of chitosanase from broth extract in a single step. A chitosanase-producing strain was isolated and identified as Bacillus cereus C-01 and used to produce chitosanases. The expanded bed adsorption conditions for chitosanase purification were optimized statistically using STREAMLINE(TM) DEAE and a homemade column (2.6 × 30.0 cm). Dependent variables were defined by the quality criteria purification factor (P) and enzyme yield to optimize the chromatographic process. Statistical analyses showed that the optimum conditions for the maximum P were 150 cm/h load flow velocity, 6.0 cm settled bed height, and 7.36 cm distributor height. Distributor height had a strong influence on the process, considerably affecting both the P and enzyme yield. Optimizing the purification variables resulted in an approximately 3.66-fold increase in the P compared with the value under nonoptimized conditions. This system is promising for the recovery of chitosanase from B. cereus C-01 and is economically viable because it promotes the reduction steps. PMID:26638991

  13. Expression, purification and crystallization of a lyssavirus matrix (M) protein

    SciTech Connect

    Assenberg, René; Delmas, Olivier; Graham, Stephen C.; Verma, Anil; Berrow, Nick; Stuart, David I.; Owens, Raymond J.; Bourhy, Hervé; Grimes, Jonathan M.

    2008-04-01

    The expression, purification and crystallization of the full-length matrix protein from three lyssaviruses is described. The matrix (M) proteins of lyssaviruses (family Rhabdoviridae) are crucial to viral morphogenesis as well as in modulating replication and transcription of the viral genome. To date, no high-resolution structural information has been obtained for full-length rhabdovirus M. Here, the cloning, expression and purification of the matrix proteins from three lyssaviruses, Lagos bat virus (LAG), Mokola virus and Thailand dog virus, are described. Crystals have been obtained for the full-length M protein from Lagos bat virus (LAG M). Successful crystallization depended on a number of factors, in particular the addition of an N-terminal SUMO fusion tag to increase protein solubility. Diffraction data have been recorded from crystals of native and selenomethionine-labelled LAG M to 2.75 and 3.0 Å resolution, respectively. Preliminary analysis indicates that these crystals belong to space group P6{sub 1}22 or P6{sub 5}22, with unit-cell parameters a = b = 56.9–57.2, c = 187.9–188.6 Å, consistent with the presence of one molecule per asymmetric unit, and structure determination is currently in progress.

  14. Improved purification and PCR amplification of DNA from environmental samples.

    PubMed

    Arbeli, Ziv; Fuentes, Cilia L

    2007-07-01

    Purification and PCR amplification procedures for DNA extracted from environmental samples (soil, compost, and river sediment) were improved by introducing three modifications: precipitation of DNA with 5% polyethylene glycol 8000 (PEG) and 0.6 M NaCl; filtration with a Sepharose 4B-polyvinylpolypyrrolidone (PVPP) spin column; and addition of skim milk (0.3% w/v) to the PCR reaction solution. Humic substances' concentration after precipitation with 5% PEG was 2.57-, 5.3-, and 78.9-fold lower than precipitation with 7.5% PEG, 10% PEG, and isopropanol, respectively. After PEG precipitation, Sepharose, PVPP and the combined (Sepharose-PVPP) column removed 92.3%, 89.5%, and 98%, respectively, of the remaining humic materials. Each of the above-mentioned modifications improved PCR amplification of the 16S rRNA gene. DNA extracted by the proposed protocol is cleaner than DNA extracted by a commercial kit. Nevertheless, the improvement of DNA purification did not improve the detection limit of atrazine degradation gene atzA. PMID:17521406

  15. EUV tools: hydrogen gas purification and recovery strategies

    NASA Astrophysics Data System (ADS)

    Landoni, Cristian; Succi, Marco; Applegarth, Chuck; Riddle Vogt, Sarah

    2015-03-01

    The technological challenges that have been overcome to make extreme ultraviolet lithography (EUV) a reality have been enormous1. This vacuum driven technology poses significant purity challenges for the gases employed for purging and cleaning the scanner EUV chamber and source. Hydrogen, nitrogen, argon and ultra-high purity compressed dry air (UHPCDA) are the most common gases utilized at the scanner and source level. Purity requirements are tighter than for previous technology node tools. In addition, specifically for hydrogen, EUV tool users are facing not only gas purity challenges but also the need for safe disposal of the hydrogen at the tool outlet. Recovery, reuse or recycling strategies could mitigate the disposal process and reduce the overall tool cost of operation. This paper will review the types of purification technologies that are currently available to generate high purity hydrogen suitable for EUV applications. Advantages and disadvantages of each purification technology will be presented. Guidelines on how to select the most appropriate technology for each application and experimental conditions will be presented. A discussion of the most common approaches utilized at the facility level to operate EUV tools along with possible hydrogen recovery strategies will also be reported.

  16. Affinity approaches in RNAi-based therapeutics purification.

    PubMed

    Pereira, Patrícia; Queiroz, João A; Figueiras, Ana; Sousa, Fani

    2016-05-15

    The recent investigation on RNA interference (RNAi) related mechanisms and applications led to an increased awareness of the importance of RNA in biology. Nowadays, RNAi-based technology has emerged as a potentially powerful tool for silencing gene expression, being exploited to develop new therapeutics for treating a vast number of human disease conditions, as it is expected that this technology can be translated onto clinical applications in a near future. This approach makes use of a large number of small (namely short interfering RNAs, microRNAs and PIWI-interacting RNAs) and long non-coding RNAs (ncRNAs), which are likely to have a crucial role as the next generation therapeutics. The commercial and biomedical interest in these RNAi-based therapy applications have fostered the need to develop innovative procedures to easily and efficiently purify RNA, aiming to obtain the final product with high purity degree, good quality and biological activity. Recently, affinity chromatography has been applied to ncRNAs purification, in view of the high specificity. Therefore, this article intends to review the biogenesis pathways of regulatory ncRNAs and also to discuss the most significant and recent developments as well as applications of affinity chromatography in the challenging task of purifying ncRNAs. In addition, the importance of affinity chromatography in ncRNAs purification is addressed and prospects for what is forthcoming are presented. PMID:26830537

  17. Rapid purification of fluorescent enzymes by ultrafiltration

    NASA Technical Reports Server (NTRS)

    Benjaminson, M. A.; Satyanarayana, T.

    1983-01-01

    In order to expedite the preparation of fluorescently tagged enzymes for histo-cyctochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

  18. Rapid purification of fluorescent enzymes by ultrafiltration

    NASA Technical Reports Server (NTRS)

    Benjaminson, M. A.; Satyanarayana, T.

    1983-01-01

    In order to expedite the preparation of fluorescently tagged enzymes for histo/cytochemistry, a previously developed method employing gel column purification was compared with a more rapid modern technique using the Millipore Immersible CX-ultrafilter. Microscopic evaluation of the resulting conjugates showed comparable products. Much time and effort is saved using the new technique.

  19. Purification of tantalum by plasma arc melting

    DOEpatents

    Dunn, Paul S.; Korzekwa, Deniece R.

    1999-01-01

    Purification of tantalum by plasma arc melting. The level of oxygen and carbon impurities in tantalum was reduced by plasma arc melting the tantalum using a flowing plasma gas generated from a gas mixture of helium and hydrogen. The flowing plasma gases of the present invention were found to be superior to other known flowing plasma gases used for this purpose.

  20. Purification of tantalum by plasma arc melting

    SciTech Connect

    Dunn, P.S.; Korzekwa, D.R.

    1999-10-26

    Purification of tantalum by plasma arc melting is disclosed. The level of oxygen and carbon impurities in tantalum was reduced by plasma arc melting the tantalum using a flowing plasma gas generated from a gas mixture of helium and hydrogen. The flowing plasma gases of the present invention were found to be superior to other known flowing plasma gases used for this purpose.

  1. Expression and Purification of Sperm Whale Myoglobin

    ERIC Educational Resources Information Center

    Miller, Stephen; Indivero, Virginia; Burkhard, Caroline

    2010-01-01

    We present a multiweek laboratory exercise that exposes students to the fundamental techniques of bacterial expression and protein purification through the preparation of sperm whale myoglobin. Myoglobin, a robust oxygen-binding protein, contains a single heme that gives the protein a reddish color, making it an ideal subject for the teaching…

  2. Sequential purification and crystal growth for the production of low cost silicon substrates. Quarterly technical progress report No. 2, 1 January 1980-31 March 1980

    SciTech Connect

    Liaw, M; Aragona, F S

    1980-05-01

    The objective of this program is to identify and develop low cost processing for fabricating large grain size polycrystalline silicon substrates. Metallurgical grade silicon (MG-Si) which is low cost and abundant for industrial usage was chosen as starting material. However, MG-Si cannot be used directly as substrates for solar cell fabrication; the further purification and recrystallization of MG-Si are needed. The conventional method of purifying MG-Si requires the conversion of Si to gaseous chlorosilanes. Chlorosilanes are purified by distallation. The purified chlorosilanes are then converted back to elemental silicon using a chemical vapor deposition process. Purified polysilicon requires recrystallization to become single crystals or large grain polycrystalline form for electronic device or solar cell applications. The techniques being studied under this program use direct methods for the purification of MG-Si. The process uses sequential steps of purification followed by crystal growth. The steps of sequential purification include: (1) leaching of MG-Si charge, 2) phase separation of non-soluble impurities from molten silicon, 3) reactive gas treatment of molten silicon, 4) liquid-liquid extraction (called slagging), and 5) impurity redistribution using ingot pulling. All the purification steps are performed in a consecutive manner using a crystal puller with the exception of step (1). The purified ingots will be in a desired ingot dimension and further recrystallization is not necessary. In this quarterly period the study of the purification by (1) reactive gas treatment, and 2) slagging was completed. Selection of reusable crucible has also been made. Progress is reported.

  3. A robust robotic high-throughput antibody purification platform.

    PubMed

    Schmidt, Peter M; Abdo, Michael; Butcher, Rebecca E; Yap, Min-Yin; Scotney, Pierre D; Ramunno, Melanie L; Martin-Roussety, Genevieve; Owczarek, Catherine; Hardy, Matthew P; Chen, Chao-Guang; Fabri, Louis J

    2016-07-15

    Monoclonal antibodies (mAbs) have become the fastest growing segment in the drug market with annual sales of more than 40 billion US$ in 2013. The selection of lead candidate molecules involves the generation of large repertoires of antibodies from which to choose a final therapeutic candidate. Improvements in the ability to rapidly produce and purify many antibodies in sufficient quantities reduces the lead time for selection which ultimately impacts on the speed with which an antibody may transition through the research stage and into product development. Miniaturization and automation of chromatography using micro columns (RoboColumns(®) from Atoll GmbH) coupled to an automated liquid handling instrument (ALH; Freedom EVO(®) from Tecan) has been a successful approach to establish high throughput process development platforms. Recent advances in transient gene expression (TGE) using the high-titre Expi293F™ system have enabled recombinant mAb titres of greater than 500mg/L. These relatively high protein titres reduce the volume required to generate several milligrams of individual antibodies for initial biochemical and biological downstream assays, making TGE in the Expi293F™ system ideally suited to high throughput chromatography on an ALH. The present publication describes a novel platform for purifying Expi293F™-expressed recombinant mAbs directly from cell-free culture supernatant on a Perkin Elmer JANUS-VariSpan ALH equipped with a plate shuttle device. The purification platform allows automated 2-step purification (Protein A-desalting/size exclusion chromatography) of several hundred mAbs per week. The new robotic method can purify mAbs with high recovery (>90%) at sub-milligram level with yields of up to 2mg from 4mL of cell-free culture supernatant. PMID:27283099

  4. Isolation and purification of kinesin from Drosophila embryos.

    PubMed

    Sigua, Robilyn; Tripathy, Suvranta; Anand, Preetha; Gross, Steven P

    2012-01-01

    Motor proteins move cargoes along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 °C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single

  5. Purification of circular DNA using benzoylated naphthoylated DEAE-cellulose.

    PubMed

    Gamper, H; Lehman, N; Piette, J; Hearst, J E

    1985-04-01

    Un-nicked circular DNA can be separated from protein, RNA, and other DNA in a simple three-step protocol consisting of exonuclease III digestion, extraction with benzoylated naphthoylated DEAE-cellulose (BND cellulose) in 1 M NaCl, and alcohol precipitation of the remaining supercoiled DNA. Exonuclease III treatment introduces single-stranded regions into contaminating linear and nicked circular DNA. This DNA, together with most RNA and protein, is adsorbed onto BND cellulose leaving form I DNA in solution. The protocol can be used to purify analytical as well as preparative amounts of supercoiled DNA. This procedure is a substitute for cesium chloride-ethidium bromide gradient ultracentrifugation and gives a comparable yield of pure form I DNA. Other classes of DNA can be isolated by changing the pretreatment step. Selective digestion of linear DNA with lambda exonuclease permits the isolation of both nicked circular and supercoiled DNA while brief heat-induced or alkali-induced denaturation leads to the recovery of rapidly reannealing DNA. In large-scale purifications, the basic protocol is usually preceded by one or more BND cellulose extractions in 1 M NaCl to remove contaminants absorbing UV or inhibiting exonuclease III. PMID:3996184

  6. Bovine kidney beta-mannosidase: purification and characterization.

    PubMed Central

    Sopher, B L; Traviss, C E; Cavanagh, K T; Jones, M Z; Friderici, K H

    1993-01-01

    Lysosomal beta-mannosidase was purified 160,000-fold in 24% yield from bovine kidney by a four-step purification procedure, which included concanavalin A-Sepharose, immunoaffinity, TSK-butyl and h.p.l.c. cation-exchange chromatography. When analysed by SDS/PAGE and detected by Coomassie Blue or silver staining, the purified enzyme preparation consists of two prominent peptides (100 and 110 kDa) and a third minor peptide (84 kDa). These three peptides are immunologically related and are consistently associated with beta-mannosidase activity in all chromatographic steps. Removal of N-linked carbohydrate from the 84, 100 and 110 kDa peptides decreases their molecular sizes to 75, 86 and 91 kDa respectively. Bovine kidneys lacking beta-mannosidase, activity, acquired from calves affected with beta-mannosidosis, do not contain detectable quantities of the three beta-mannosidase peptides, as judged by monoclonal- and polyclonal-antibody reactivity. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8424779

  7. Micromachine Wedge Stepping Motor

    SciTech Connect

    Allen, J.J.; Schriner, H.K.

    1998-11-04

    A wedge stepping motor, which will index a mechanism, has been designed and fabricated in the surface rnicromachine SUMMiT process. This device has demonstrated the ability to index one gear tooth at a time with speeds up to 205 teeth/see. The wedge stepper motor has the following features, whi:h will be useful in a number of applications. o The ability to precisely position mechanical components. . Simple pulse signals can be used for operation. o Only 2 drive signals are requixed for operation. o Torque and precision capabilities increase with device size . The device to be indexed is restrained at all times by the wedge shaped tooth that is used for actuation. This paper will discuss the theory of operation and desi=m of the wedge stepping motor. The fabrication and testing of I he device will also be presented.

  8. The effectiveness of components of University of Wisconsin solution in improving human pancreatic islet purification.

    PubMed

    Robertson, G S; Chadwick, D R; Davies, J; Rose, S; Contractor, H; James, R F; Bell, P R; London, N J

    1994-02-01

    The purification of human pancreatic islets before transplantation relies on the density-dependent separation of islets from exocrine fragments after collagenase digestion of the donor pancreas. The results vary among pancreases despite increasing automation of the digestion and purification processes, reflecting variations in the overlapping densities of islets and contaminating exocrine tissue. Hypothermic storage of both the pancreas and the pancreatic digest alters cell volumes and tissue densities, thereby affecting islet purification. By biochemical analysis of the isopycnic distribution of islets and exocrine tissue fragments from 23 human pancreases on linear continuous density gradients, the effect of various solutions for cold storage of pancreatic digest was studied. The use of the University of Wisconsin cold storage solution, which resulted in a significant decrease in digest volume (P = 0.006) and increase in the densities of both exocrine tissue (P = 0.001) and islets (P = 0.005), produced a significant improvement in islet purity compared with tissue culture medium (P = 0.035), predominantly due to the inclusion of a colloid, which increased the difference in density between exocrine tissue and islets. The addition of large molecular weight cellular impermeants without alteration in the concentration of permeable anions produced no effect. The results of this study support the concept that the use of solutions that minimize cell swelling throughout the process of islet purification would result in significant improvements in density-dependent islet separation, and that such solutions should contain a colloid. PMID:8108869

  9. Enhanced method for microbial community DNA extraction and purification from agricultural yellow loess soil.

    PubMed

    Kathiravan, Mathur Nadarajan; Gim, Geun Ho; Ryu, Jaewon; Kim, Pyung Il; Lee, Chul Won; Kim, Si Wouk

    2015-11-01

    In this study, novel DNA extraction and purification methods were developed to obtain high-quantity and reliable quality DNA from the microbial community of agricultural yellow loess soil samples. The efficiencies of five different soil DNAextraction protocols were evaluated on the basis of DNA yield, quality and DNA shearing. Our suggested extraction method, which used CTAB, EDTA and cell membrane lytic enzymes in the extraction followed by DNA precipitation using isopropanol, yielded a maximum DNA content of 42.28 ± 5.59 µg/g soil. In addition, among the five different purification protocols, the acid-treated polyvinyl polypyrrolidone (PVPP) spin column purification method yielded high-quality DNA and recovered 91% of DNA from the crude DNA. Spectrophotometry revealed that the ultraviolet A 260/A 230 and A 260/A 280 absorbance ratios of the purified DNA were 1.82 ± 0.03 and 1.94 ± 0.05, respectively. PCR-based 16S rRNA amplification showed clear bands at ~1.5 kb with acid-treated PVPP-purified DNA templates. In conclusion, our suggested extraction and purification protocols can be used to recover high concentration, high purity, and high-molecular-weight DNA from clay and silica-rich agricultural soil samples. PMID:26502961

  10. Effect of purification pretreatment on the recovery of magnetite from waste ferrous sulfate

    NASA Astrophysics Data System (ADS)

    Yu, Wang; Peng, Ying-lin; Zheng, Ya-jie

    2016-08-01

    The present study was conducted to elucidate the influence of impurities in waste ferrous sulfate on its recovery of magnetite. Ferrous sulfate solution was purified by the addition of NaOH solution to precipitate impurities, and magnetite was recovered from ferrous sulfate solution without and with purification pretreatment. Calcium hydroxide was added to the solution of ferrous sulfate as a precipitator. A mixed product of magnetite and gypsum was subsequently obtained by air oxidation and heating. Wet-milling was performed prior to magnetic separation to recover magnetite from the mixed products. The results show that with the purification pretreatment, the grade of iron in magnetite concentrate increased from 62.05% to 65.58% and the recovery rate of iron decreased from 85.35% to 80.35%. The purification pretreatment reduced the conglutination between magnetite and gypsum, which favors their subsequent magnetic separation. In summary, a higher-grade magnetite with a better crystallinity and a larger particle size of 2.35 μm was obtained with the purification pretreatment.

  11. Single step purification procedure for the rapid separation of equine leucocytes.

    PubMed

    Sedgwick, A D; Morris, T; Russell, B A; Lees, P

    1986-11-01

    Percoll gradients have been used to separate relatively pure populations of viable equine polymorphonuclear (PMN) and mononuclear (MN) cells. In preliminary studies, a continuous density gradient of 70% Percoll solution was used to separate two distinct leucocyte-rich bands. After measurement of the density of each band on the continuous gradient, discontinuous Percoll gradients, using 60% and 75% Percoll solutions, were used to provide a rapid means of separating PMN and MN cells. The yield of viable cells per ml of blood was 3.0 X 10(6) and 3.2 X 10(6) for MN and PMN cells, respectively. Corresponding values for recovery were 45% and 72%. The purity was 94% for PMNs and 99% for MNs. PMID:3798735

  12. Purification and characterization of an extracellular cellulase from Anoxybacillus gonensis O9 isolated from geothermal area in Turkey.

    PubMed

    Genc, Berna; Nadaroglu, Hayrunnisa; Adiguzel, Ahmet; Baltaci, Ozkan

    2015-11-01

    In the present study, cellulase was purified and characterized from Anoxybacillus gonensis (Gen bank Number: KM596794) which was isolated and characterized from Agri Diyadin Hot spring. It was found to synthesize cellulase which had a wide range of industrial applications. Twenty four-hour-cultured bacteria induced cellulase production and specific activities during the purification steps were 1.47, 81.06 and 109.4 EU mg(-1) protein at crude extract, ammonium sulphate precipitated and DEAE-Sephadex purification steps. The highest enzyme activity was observed at 50°C and the optimum range of pH was 3-10. Molecular weight of enzyme was determined approximately 40kDa. The kinetic parameters of cellulase against carboxymethylcellulose (CMC) were 153.4 pmol min(-1) mg for Vmax and 0.46mM for Km. Among effectors of the enzyme, Zn2+, Ca2+, Co2+ and EDTA decreased enzyme activity. PMID:26688967

  13. New photolithography stepping machine

    SciTech Connect

    Hale, L.; Klingmann, J.; Markle, D.

    1995-03-08

    A joint development project to design a new photolithography steeping machine capable of 150 nanometer overlay accuracy was completed by Ultratech Stepper and the Lawrence Livermore National Laboratory. The principal result of the project is a next-generation product that will strengthen the US position in step-and-repeat photolithography. The significant challenges addressed and solved in the project are the subject of this report. Design methods and new devices that have broader application to precision machine design are presented in greater detail while project specific information serves primarily as background and motivation.

  14. Stepped sinewave inverter

    NASA Astrophysics Data System (ADS)

    Appelbaum, J.; Gabbay, D.

    1984-11-01

    A stepped sinewave dc/ac inverter was analyzed for an inductive load with respect to load current and voltage, harmonics, power factor, and efficiency. This special inverter of high efficiency and low harmonic content is constructed by synthesizing the sinusoidal output by discrete voltage sources, such as storage batteries, solar cell, etc., with electronic switching of the sources at specific time intervals. The switching times are determined for the condition of minimum distortion of the synthesized wave. A 50 W inverter was built and tested to demonstrate this approach.

  15. Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation.

    PubMed

    Ruff, Jürgen; Denger, Karin; Cook, Alasdair M

    2003-01-15

    The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum. PMID:12358600

  16. Purification and characterization of a fish granzymeA involved in cell-mediated immunity.

    PubMed

    Matsuura, Yuta; Yabu, Takeshi; Shiba, Hajime; Moritomo, Tadaaki; Nakanishi, Teruyuki

    2016-07-01

    Granzymes are serine proteases involved in the induction of cell death against non-self cells. The enzymes differ in their primary substrate specificity and have one of four hydrolysis activities: tryptase, Asp-ase, Met-ase and chymase. Although granzyme genes have been isolated from several fishes, evidence for their involvement in cytotoxicity has not yet been reported. In the present study, we attempted to purify and characterize a fish granzyme involved in cytotoxicity using ginbuna crucian carp. The cytotoxicity of leukocytes was significantly inhibited by the serine protease inhibitor ''3, 4-dichloroisocoumarin''. In addition, we found that granzymeA-like activity (hydrolysis of Z-GPR-MCA) was inhibited by the same inhibitor and significantly enhanced by allo-antigen stimulation in vivo. Proteins from leukocyte extracts were subjected to two steps of chromatographic purification using benzamidine-Sepharose and SP-Sepharose. The molecular weight of the purified enzyme was estimated to be 26,900 Da by SDS-PAGE analysis. The purified enzyme displayed a Km of 220 μM, a Kcat of 21.7 sec(-1) and a Kcat/Km of 98,796 sec(-1) M(-1) with an optimal pH of 9.5 for the Z-GPR-MCA substrate. The protease was totally inhibited by serine protease inhibitors and showed granzymeA-like substrate specificity. Therefore, we conclude that the purified enzyme belongs to the mammalian granzymeA (EC 3.4.21.78) and appears to be involved in cytotoxicity in fish. PMID:26872543

  17. World-to-digital-microfluidic interface enabling extraction and purification of RNA from human whole blood.

    PubMed

    Jebrail, Mais J; Sinha, Anupama; Vellucci, Samantha; Renzi, Ronald F; Ambriz, Cesar; Gondhalekar, Carmen; Schoeniger, Joseph S; Patel, Kamlesh D; Branda, Steven S

    2014-04-15

    Digital microfluidics (DMF) is a powerful technique for simple and precise manipulation of microscale droplets of fluid. This technique enables processing and analysis of a wide variety of samples and reagents and has proven useful in a broad range of chemical, biological, and medical applications. Handling of "real-world" samples has been a challenge, however, because typically their volumes are greater than those easily accommodated by DMF devices and contain analytes of interest at low concentration. To address this challenge, we have developed a novel "world-to-DMF" interface in which an integrated companion module drives the large-volume sample through a 10 μL droplet region on the DMF device, enabling magnet-mediated recovery of bead-bound analytes onto the device as they pass through the region. To demonstrate its utility, we use this system for extraction of RNA from human whole blood lysates (110-380 μL) and further purification in microscale volumes (5-15 μL) on the DMF device itself. Processing by the system was >2-fold faster and consumed 12-fold less reagents, yet produced RNA yields and quality fully comparable to conventional preparations and supporting qRT-PCR and RNA-Seq analyses. The world-to-DMF system is designed for flexibility in accommodating different sample types and volumes, as well as for facile integration of additional modules to enable execution of more complex protocols for sample processing and analysis. As the first technology of its kind, this innovation represents an important step forward for DMF, further enhancing its utility for a wide range of applications. PMID:24479881

  18. Sulphoacetaldehyde acetyltransferase yields acetyl phosphate: purification from Alcaligenes defragrans and gene clusters in taurine degradation.

    PubMed Central

    Ruff, Jürgen; Denger, Karin; Cook, Alasdair M

    2003-01-01

    The facultatively anaerobic bacterium Alcaligenes defragrans NKNTAU was found to oxidize taurine (2-aminoethanesulphonate) with nitrate as the terminal electron acceptor. Taurine was transaminated to 2-sulphoacetaldehyde. This was not converted into sulphite and acetate by a "sulphoacetaldehyde sulpho-lyase" (EC 4.4.1.12), but into sulphite and acetyl phosphate, which was identified by three methods. The enzyme, which required the addition of phosphate, thiamin diphosphate and Mg(2+) ions for activity, was renamed sulphoacetaldehyde acetyltransferase (Xsc; EC 2.3.1.-). Inducible Xsc was expressed at high levels, and a three-step 11-fold purification yielded an essentially homogeneous soluble protein, which was a homotetramer in its native form; the molecular mass of the subunit was found to be between about 63 kDa (SDS/PAGE) and 65.3 kDa (matrix-assisted laser-desorption ionization-time-of-flight MS). The N-terminal and two internal amino acid sequences were determined, and PCR primers were generated. The xsc gene was amplified and sequenced; the derived molecular mass of the processed protein was 65.0 kDa. The downstream gene presumably encoded the inducible phosphate acetyltransferase (Pta) found in crude extracts. The desulphonative enzymes ("EC 4.4.1.12") from Achromobacter xylosoxidans NCIMB 10751 and Desulfonispora thiosulfatigenes GKNTAU were shown to be Xscs. We detected at least three subclasses of xsc in Proteobacteria and in Gram-positive bacteria, and they comprised a distinct group within the acetohydroxyacid synthase supergene family. Genome sequencing data revealed xsc genes in Burkholderia fungorum (80% sequence identity) and Sinorhizobium meliloti (61%) with closely linked pta genes. Different patterns of regulation for the transport and dissimilation of taurine were hypothesized for S. meliloti and B. fungorum. PMID:12358600

  19. Expression, purification, and crystallization of Schizosaccharomyces pombe eIF2B.

    PubMed

    Kashiwagi, Kazuhiro; Shigeta, Tomoaki; Imataka, Hiroaki; Ito, Takuhiro; Yokoyama, Shigeyuki

    2016-03-01

    Tight control of protein synthesis is necessary for cells to respond and adapt to environmental changes rapidly. Eukaryotic translation initiation factor (eIF) 2B, the guanine nucleotide exchange factor for eIF2, is a key target of translation control at the initiation step. The nucleotide exchange activity of eIF2B is inhibited by the stress-induced phosphorylation of eIF2. As a result, the level of active GTP-bound eIF2 is lowered, and protein synthesis is attenuated. eIF2B is a large multi-subunit complex composed of five different subunits, and all five of the subunits are the gene products responsible for the neurodegenerative disease, leukoencephalopathy with vanishing white matter. However, the overall structure of eIF2B has remained unresolved, due to the difficulty in preparing a sufficient amount of the eIF2B complex. To overcome this problem, we established the recombinant expression and purification method for eIF2B from the fission yeast Schizosaccharomyces pombe. All five of the eIF2B subunits were co-expressed and reconstructed into the complex in Escherichia coli cells. The complex was successfully purified with a high yield. This recombinant eIF2B complex contains each subunit in an equimolar ratio, and the size exclusion chromatography analysis suggests it forms a heterodecamer, consistent with recent reports. This eIF2B increased protein synthesis in the reconstituted in vitro human translation system. In addition, disease-linked mutations led to subunit dissociation. Furthermore, we crystallized this functional recombinant eIF2B, and the crystals diffracted to 3.0 Å resolution. PMID:27023709

  20. High-level secretory expression and purification of unhydroxylated human collagen α1(III) chain in Pichia pastoris GS115.

    PubMed

    Li, Linbo; Fan, Daidi; Ma, Xiaoxuan; Deng, Jianjun; He, Jing

    2015-01-01

    Recombinant collagen and gelatin have been applied in biomedical materials field because of products from genetically engineered microorganisms with improved safety, traceability, reproducibility, and homogeneous quality. To obtain high-level secretory expression of single-chain full-length human collagen α1(III) chain (COL3A1) without the N and C telopeptides, the cDNA coding for the human COL3A1 gene was cloned into the secretory expression vector pPIC9K and integrated into Pichia pastoris GS115. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis of culture supernatant from the recombinant methylotrophic yeast suggested that the unhydroxylated recombinant human COL3A1 (rhCOL3A1) was secreted into the culture medium, and exhibited an apparent molecular mass of approximately 130 kDa, which is 1.4 times higher than the theoretical one. Finally, the unhydroxylated rhCOL3A1 was purified to greater than 90% purity using a four-step approach. In addition, methylthiazolydiphenyl-tetrazolium bromide experiments indicated that low concentration of rhCOL3A1 could promote Baby hamster kidney cell (BHK21) proliferation effectively. The production and purification of rhCOL3A1 described in this study offer a new method for obtaining high level of rhCOL3A1 in relatively pure form, which is suitable for biomedical materials application. PMID:25231012

  1. Development of a novel and efficient cell culture flocculation process using a stimulus responsive polymer to streamline antibody purification processes.

    PubMed

    Kang, Yun Kenneth; Hamzik, James; Felo, Michael; Qi, Bo; Lee, Julia; Ng, Stanley; Liebisch, Gregory; Shanehsaz, Behnam; Singh, Nripen; Persaud, Kris; Ludwig, Dale L; Balderes, Paul

    2013-11-01

    Recent advances in mammalian cell culture processes have significantly increased product titers, but have also resulted in substantial increases in cell density and cellular debris as well as process and product related impurities. As such, with improvements in titer, corresponding improvements in downstream processing are essential. In this study we have developed an alternative antibody harvest process that incorporates flocculation using a novel stimulus responsive polymer, benzylated poly(allylamine), followed by depth filtration. As tested on multiple antibodies, this process demonstrates high process yield, improved clearance of cells and cell debris, and efficient reduction of aggregates, host cell proteins (HCP) and DNA. A wide operating window was established for this novel flocculation process through design of experiments condition screening and optimization. Residual levels of impurities in the Protein A eluate were achieved that potentially meet requirements of drug substance and thus alleviate the burden for further impurities removal in subsequent chromatography steps. In addition, efficient clearance of residual polymer was demonstrated using a fluorescence tagged polymer in the presence of a stimulus reagent. The mechanism of HCP and aggregates removal during flocculation was also explored. This novel and efficient process can be easily integrated into current mAb purification platforms, and may overcome downstream processing challenges. PMID:23740533

  2. Distillation and purification of symmetric entangled Gaussian states

    SciTech Connect

    Fiurasek, Jaromir

    2010-10-15

    We propose an entanglement distillation and purification scheme for symmetric two-mode entangled Gaussian states that allows to asymptotically extract a pure entangled Gaussian state from any input entangled symmetric Gaussian state. The proposed scheme is a modified and extended version of the entanglement distillation protocol originally developed by Browne et al. [Phys. Rev. A 67, 062320 (2003)]. A key feature of the present protocol is that it utilizes a two-copy degaussification procedure that involves a Mach-Zehnder interferometer with single-mode non-Gaussian filters inserted in its two arms. The required non-Gaussian filtering operations can be implemented by coherently combining two sequences of single-photon addition and subtraction operations.

  3. Purification and characterization of glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) from pea seeds.

    PubMed

    Gani, Zahid; Boradia, Vishant Mahendra; Raghu Ram, Janaki; Suryavanshi, Prashant Mohan; Patil, Pravinkumar; Kumar, Santosh; Singh, Ranvir; Raje, Manoj; Raje, Chaaya Iyengar

    2016-11-01

    Glyceraldehyde-3-phosphate dehydrogenase [GAPDH, NAD + oxidoreductase (phosphorylating) 1.2.1.12] catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate coupled with the reduction of NAD(+) to NADH. In addition to its role in glycolysis, this enzyme has numerous alternate functions, in both prokaryotes and eukaryotes. In plants, additional functions have been reported from multiple species including Pisum sativum. A recent study has identified that GAPDH may play an important role in seed ageing and programmed cell death. Despite this the existing purification protocols are almost 40 years old, and only partial characterization of the enzyme has been reported. In the current study, we report a modified method for purification of enzymatically active pea seed GAPDH along with the characterization of the enzyme. Using 2D gel electrophoresis our study also demonstrates that pea seeds contain four isoforms of NAD(+) dependent GAPDH. PMID:27389468

  4. Purification of selenoprotein P from human plasma using immunoaffinity chromatography

    SciTech Connect

    Aakesson, B.; Bellew, T.; Burk, R.F. )

    1991-03-11

    Selenoprotein P was purified from rat plasma using immunoaffinity chromatography. The same approach was used with human plasma. HepG2 cells were labeled with {sup 75}Se. The labeled medium, containing proteins secreted by the cells, was added to human plasma and the {sup 75}Se was used as a marker for {gt}1,000-fold purification of the major {sup 75}Se-containing protein. This material was used to produce 2 monoclonal antibodies. In a competitive assay, human plasma, but not plasma from 5 other species, inhibited binding of {sup 75}Se by these 2 antibodies. The antibodies were coupled to agarose and columns were made. Human plasma was processed in 2 steps. Step 1 was an antibody column and step 2 was a heparin-agarose column. SDS-PAGE demonstrated bands at 61 and 55 kDa. Both bands stained with PAS. Amino acid analysis of carboxymethylated material indicated that selenocysteine was {gt}1% of the total amino acids. N-terminal sequencing revealed a strong similarity to rat selenoprotein P. Immunodepleted human plasma and control plasma were chromatographed on Sephacryl S200 and selenium was measured in the eluted fractions. Immunodepletion removed one-third of the selenium. The elution pattern of control plasma revealed a broad peak of selenium just ahead of and including the albumin peak. Most of this peak was absent from the immunodepleted serum and a graph of the difference between the 2 chromatograms was a single peak of selenium well separated from the albumin peak.

  5. Evaluation of the purification capacity of nine portable, small-scale water purification devices.

    PubMed

    Hörman, A; Rimhanen-Finne, R; Maunula, L; von Bonsdorff, C H; Rapala, J; Lahti, K; Hänninen, M L

    2004-01-01

    A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters. PMID:15318506

  6. SPAR-H Step-by-Step Guidance

    SciTech Connect

    W. J. Galyean; A. M. Whaley; D. L. Kelly; R. L. Boring

    2011-05-01

    This guide provides step-by-step guidance on the use of the SPAR-H method for quantifying Human Failure Events (HFEs). This guide is intended to be used with the worksheets provided in: 'The SPAR-H Human Reliability Analysis Method,' NUREG/CR-6883, dated August 2005. Each step in the process of producing a Human Error Probability (HEP) is discussed. These steps are: Step-1, Categorizing the HFE as Diagnosis and/or Action; Step-2, Rate the Performance Shaping Factors; Step-3, Calculate PSF-Modified HEP; Step-4, Accounting for Dependence, and; Step-5, Minimum Value Cutoff. The discussions on dependence are extensive and include an appendix that describes insights obtained from the psychology literature.

  7. Coexistence of meandering and bunching of steps on vicinal surfaces

    NASA Astrophysics Data System (ADS)

    Yu, Yan-Mei; Liu, Bang-Gui

    2006-01-01

    We simulate morphology and its evolution of vicinal surfaces in epitaxy by using the phase-field model. For usual parameters of Cu vicinal surfaces, a pure in-phase meandering pattern consistent to the experimental images is obtained. Nevertheless, vicinal surfaces grow into more complex hierarchy for a small kink energy. In addition to step meandering, step bunching happens due to competition of local step fluctuation and interlayer Ehrlich-Schwoebel barrier in the presence of the step meandering. This implies that step meandering and step bunching can coexist in some growing epitaxial vicinal surfaces.

  8. Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin.

    PubMed Central

    Gabay, J E; Blake, M; Niles, W D; Horwitz, M A

    1985-01-01

    We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins. Images PMID:2579942

  9. Using Mass Transport to Guide the Purification of Small Molecule Organic Semiconductors via Sublimation

    NASA Astrophysics Data System (ADS)

    Morgan, Nathan T.; Zhang, Yi; Grandbois, Matthew L.; Bell, Bruce M.; Holmes, Russell J.; Cussler, E. L.

    2015-03-01

    Organic electronic materials have garnered considerable commercial attention for next generation display and solid-state lighting applications. Widespread adoption of these technologies is slowed by considerable production costs, partially due to an expensive purification step. This work explores the current method of industrial purification, thermal gradient sublimation, in order to isolate the fundamental mechanisms limiting sublimation rate and controlling product deposition. For the archetypical hole transport materials, N,N'-bis(naphthalen-1-yl)-N,N'-bis(phenyl)-benzidine (NPD) and 4,4',4''-tris(carbazol-9-yl) triphenylamine (TCTA), a combination of viscous flow and physical vapor deposition are shown to be rate-limiting at constant sublimation temperature. Surprisingly, diffusion within the solid feed, reaction at the feed particle surface, and mass transfer within the bed of feed particles are not rate limiting in the case. This mechanism is different from that which is observed in many industrial sublimation systems. These results can be used to guide the design and operation of future large-scale purification systems, which are critical for the widespread adoption of organic optoelectronic devices.

  10. Chromatographic purification of equine immunoglobulin G F(ab)2 from plasma.

    PubMed

    Fernandes, Aldon; Kaundinya, John Oswald; Daftary, Gautam; Saxena, Lalit; Banerjee, Subhasis; Pattnaik, Priyabrata

    2008-12-01

    The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab)(2), because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab)(2) from horse serum. F(ab)(2) was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab)(2) was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab)(2) was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab)(2) was biologically functional and was found to show a potency of 1500 IU ml(-1). Comparative analysis of the purity with commercially available F(ab)(2) by HPLC analysis and SDS-PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media. PMID:19008160

  11. Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe.

    PubMed

    Ball, H L; Bertolini, G; Mascagni, P

    1995-01-01

    The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin-agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC. PMID:9223007

  12. Isolation and purification of blood group antigens using immuno-affinity chromatography on short monolithic columns.

    PubMed

    Mönster, Andrea; Hiller, Oliver; Grüger, Daniela; Blasczyk, Rainer; Kasper, Cornelia

    2011-02-01

    Monolithic columns have gained increasing attention as stationary phases for the separation of biomolecules and biopharmaceuticals. In the present work the performance of monolithic convective interaction media (CIM(®)) chromatography for the purification of blood group antigens was established. The proteins employed in this study are derived from blood group antigens Knops, JMH and Scianna, equipped both with a His-tag and with a V5-tag by which they can be purified. In a first step a monoclonal antibody directed against the V5-tag was immobilized on a CIM(®) Disk with epoxy chemistry. After this, the immobilized CIM(®) Disk was used in immuno-affinity chromatography to purify the three blood group antigens from cell culture supernatant. Up-scaling of the applied technology was carried out using CIM(®) Tubes. In comparison to conventional affinity chromatography, blood group antigens were also purified via His-tag using a HiTrap(®) metal-affinity column. The two purifications have been compared regarding purity, yield and purification speed. Using the monolithic support, it was possible to isolate the blood group antigens with a higher flow rate than using the conventional bed-packed column. PMID:21194702

  13. Purification of HIV-1 gag virus-like particles and separation of other extracellular particles.

    PubMed

    Steppert, Petra; Burgstaller, Daniel; Klausberger, Miriam; Berger, Eva; Aguilar, Patricia Pereira; Schneider, Tobias A; Kramberger, Petra; Tover, Andres; Nöbauer, Katharina; Razzazi-Fazeli, Ebrahim; Jungbauer, Alois

    2016-07-15

    Enveloped virus-like particles (VLPs) are increasingly used as vaccines and immunotherapeutics. Frequently, very time consuming density gradient centrifugation techniques are used for purification of VLPs. However, the progress towards optimized large-scale VLP production increased the demand for fast, cost efficient and scale able purification processes. We developed a chromatographic procedure for purification of HIV-1 gag VLPs produced in CHO cells. The clarified and filtered cell culture supernatant was directly processed on an anion-exchange monolith. The majority of host cell impurities passed through the column, whereas the VLPs were eluted by a linear or step salt gradient; the major fraction of DNA was eluted prior to VLPs and particles in the range of 100-200nm in diameter could be separated into two fractions. The earlier eluted fraction was enriched with extracellular particles associated to exosomes or microvesicles, whereas the late eluting fractions contained the majority of most pure HIV-1 gag VLPs. DNA content in the exosome-containing fraction could not be reduced by Benzonase treatment which indicated that the DNA was encapsulated. Many exosome markers were identified by proteomic analysis in this fraction. We present a laboratory method that could serve as a basis for rapid downstream processing of enveloped VLPs. Up to 2000 doses, each containing 1×10(9) particles, could be processed with a 1mL monolith within 47min. The method compared to density gradient centrifugation has a 220-fold improvement in productivity. PMID:27286649

  14. Ecologically clean and raw-resources-saving technology using photonic purification of fibers

    NASA Astrophysics Data System (ADS)

    Belyshev, Boris E.; Protasova, Valentina A.; Shepelev, Andrey V.; Kalinin, Yuri A.

    1995-04-01

    The elaborated technology of laseral optical pumping is successfully used for the purification of fibers from impurities. This is the novel and effective application of pulsed power. The higher powerful and technological advantages for purification of wool from vegetable impurities are presented in this paper. The aim of using light irradiation is almost complete cleaning of light wool from dark vegetable admixtures, dust and brand at the early steps (carding worsted blends) of technological process of producing fine yarns. Stabilizing all mechanical and chemical-technological processes following light radiation purification and increasing technical-economic parameters of production is achieved in this case as well. The physical essence of light irradiation wool cleaning is based upon different optical and thermal properties of light fibers on the one hand, and of dark admixtures-fractions of vegetable matter and burrs, dust and brand-on the other hand. The difference of coefficients of light irradiation by fibers and impurities is of great importance. The 500-1100 nm spectral range has a selective impact on the fibers and impurities.

  15. Affinity chromatography purification of angiotensin II reactor using photoactivable biotinylated probes

    SciTech Connect

    Marie, J.; Seyer, R.; Lombard, C.; Desarnaud, F.; Aumelas, A.; Jard, A.; Bonnafous, J.C. )

    1990-09-25

    The authors have developed biotinylated photoactivable probes that are suitable for covalent labeling of angiotensin II (AII) receptors and the subsequent purification of covalent complexes through immobilized avidin or streptavidin. One of these probes, biotin-NH(CH{sub 2}){sub 2}SS(CH{sub 2}){sub 2}CO-(Ala{sup 1}, Phe(4N{sub 3}){sup 8})AII, which contains a cleavage disulfide bridge in its spacer arm and which displays, in its radioiodinated form, very high affinity for AII receptors (K{sub d}{approximately}1 nM), proved to be suitable for indirect affinity chromatography of rate liver receptor with facilitated recovery from avidin gels by use of reducing agents. This constituted the central step of an efficient partial purification scheme involving hydroxylapatite chromatography, streptavidin chromatography, and thiopropyl-Sepharose chromatography. SDS-PAGE analysis and autoradiography established the identity of the purified entity (molecular weight 65K) as the AII receptor. Possible ways of completing purification to homogeneity and extrapolation of the protocols to a preparative scale are discussed, as well as the potential contribution of our new probes to the study of the structural properties of angiotensin receptors.

  16. Purification of nanoparticles by hollow fiber diafiltration

    NASA Astrophysics Data System (ADS)

    Veeken, J.

    2012-09-01

    Hollow Fiber Diafiltration (Hollow Fiber Tangential Flow Filtration) is an efficient and rapid alternative to traditional methods of nanoparticle purification such as ultracentrifugation, stirred cell filtration, dialysis or chromatography. Hollow Fiber Diafiltration can be used to purify a wide range of nanoparticles including liposomes, colloids, magnetic particles and nanotubes. Hollow Fiber Diafiltration is a membrane based method where pore size determines the retention or transmission of solution components. It is a flow process where the sample is gently circulated through a tubular membrane. With controlled replacement of the permeate or (dialysate), pure nanoparticles can be attained. Hollow Fiber Diafiltration can be directly scaled up from R&D volumes to production. By adding more membrane fibers and maintaining the operating parameters, large volumes can be processed in the same time with the same pressure, and flow dynamics as bench-scale volumes. Keywords: hollow fiber, Diafiltration, filtration, purification, tangential flow filtration.

  17. Purification of Logic-Qubit Entanglement

    NASA Astrophysics Data System (ADS)

    Zhou, Lan; Sheng, Yu-Bo

    2016-07-01

    Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network.

  18. Purification of Logic-Qubit Entanglement.

    PubMed

    Zhou, Lan; Sheng, Yu-Bo

    2016-01-01

    Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network. PMID:27377165

  19. Purification of Logic-Qubit Entanglement

    PubMed Central

    Zhou, Lan; Sheng, Yu-Bo

    2016-01-01

    Recently, the logic-qubit entanglement shows its potential application in future quantum communication and quantum network. However, the entanglement will suffer from the noise and decoherence. In this paper, we will investigate the first entanglement purification protocol for logic-qubit entanglement. We show that both the bit-flip error and phase-flip error in logic-qubit entanglement can be well purified. Moreover, the bit-flip error in physical-qubit entanglement can be completely corrected. The phase-flip in physical-qubit entanglement error equals to the bit-flip error in logic-qubit entanglement, which can also be purified. This entanglement purification protocol may provide some potential applications in future quantum communication and quantum network. PMID:27377165

  20. Dengue virus growth, purification, and fluorescent labeling.

    PubMed

    Zhang, Summer; Chan, Kuan Rong; Tan, Hwee Cheng; Ooi, Eng Eong

    2014-01-01

    The early events of the dengue virus life cycle involve virus binding, internalization, trafficking, and fusion. Fluorescently labeled viruses can be used to visualize these early processes. As dengue virus has 180 identical copies of the envelope protein attached to the membrane surface and is surrounded by a lipid membrane, amine-reactive (Alexa Fluor) or lipophilic (DiD) dyes can be used for virus labeling. These dyes are highly photostable and are ideal for studies involving cellular uptake and endosomal transport. To improve virus labeling efficiency and minimize the nonspecific labeling of nonviral proteins, virus concentration and purification precede fluorescent labeling of dengue viruses. Besides using these viruses for single-particle tracking, DiD-labeled viruses can also be used to distinguish serotype-specific from cross-neutralizing antibodies. Here the details of virus concentration, purification, virus labeling, applications, and hints of troubleshooting are described. PMID:24696327

  1. A two-step, formal [4 + 2] approach toward piperidin-4-ones via Au catalysis.

    PubMed

    Cui, Li; Peng, Yu; Zhang, Liming

    2009-06-24

    An efficient, formal [4 + 2] synthesis of synthetically valuable piperidin-4-ones from secondary amines in two steps has been achieved via a key gold catalysis without the purification of tertiary amine intermediates. This reaction is selective toward the less-substituted alkyl group and shows moderate to excellent diastereoselectivities. Its synthetic potential in alkaloid synthesis is demonstrated in a highly diastereoselective synthesis of (+/-)-cermizine C. PMID:19492799

  2. A one-step bioprocess for production of high-content fructo-oligosaccharides from inulin by yeast.

    PubMed

    Wang, Da; Li, Fu-Li; Wang, Shi-An

    2016-10-20

    Commercial fructo-oligosaccharides (FOS) are predominantly produced from sucrose by transfructosylation process that presents a maximum theoretical yield below 0.60gFOSgSucrose(-1). To obtain high-content FOS, costly purification is generally employed. Additionally, high-content FOS can be produced from inulin by using endo-inulinases. However, commercial endo-inulinases have not been extensively used in scale-up production of FOS. In the present study, a one-step bioprocess that integrated endo-inulinase production, FOS fermentation, and non-FOS sugars removal into one reactor was proposed to produce high-content FOS from inulin. The bioprocess was implemented by a recombinant yeast strain JZHΔS-TSC, in which a heterologous endo-inulinase gene was expressed and the inherent invertase gene SUC2 was disrupted. FOS fermentation at 40°C from 200g/L chicory inulin presented the maximun titer, yield, and productivity of 180.2±0.8g/L, 0.9gFOSgInulin(-1), and 7.51±0.03g/L/h, respectively. This study demonstrated that the one-step bioprocess was simple and highly efficient. PMID:27474673

  3. Purification of cytochrome P-450 enzymes.

    PubMed

    Bell-Parikh, L C; Hosea, N A; Martin, M V; Guengerich, F P

    2002-01-01

    Among the liver P-450 xenobiotic-metabolizing enzymes, P450-2E1 is of interest because of its activation of potent carcinogens, and P-450 1A2 is of interest because of its role in oxidation of drugs and carcinogens. This unit describes column chromatography protocols for purification of recombinant forms of these enzymes expressed in a bacterial expression system. PMID:23045082

  4. Identification, Purification, and Characterization of Staphylococcal Superantigens.

    PubMed

    Merriman, Joseph A; Schlievert, Patrick M

    2016-01-01

    Purifying natively produced staphylococcal superantigens is an important process in the study of these proteins, as many common methods of protein purification are affected by staphylococcal protein A contamination. Here, we describe a proven approach for identifying superantigens in vitro as well as for purifying novel superantigens both in His-tagged and native forms using modern genetic tools coupled with thin-layer isoelectric focusing. PMID:26676034

  5. Purification of metal-organic framework materials

    SciTech Connect

    Farha, Omar K.; Hupp, Joseph T.

    2012-12-04

    A method of purification of a solid mixture of a metal-organic framework (MOF) material and an unwanted second material by disposing the solid mixture in a liquid separation medium having a density that lies between those of the wanted MOF material and the unwanted material, whereby the solid mixture separates by density differences into a fraction of wanted MOF material and another fraction of unwanted material.

  6. APPARATUS FOR THE PURIFICATION OF CALCIUM

    DOEpatents

    Burnett, R.L.

    1953-08-25

    The present patent claims and describes an apparatus adapted to carry out a new process for the purification of calcium containing an alkali metal as impurity. The process consists of distilling the impure caldium in the presence of an inert gas and at a reduced pressure, condensing substantially pure calcium on a condensing surface of iron or a ferrous alloy and condensing the alkali metal on a separate surface, the two condensing surfaces being maintained at suitable temperatures by separate cooling means.

  7. Purification of metal-organic framework materials

    SciTech Connect

    Farha, Omar K.; Hupp, Joseph T.

    2015-06-30

    A method of purification of a solid mixture of a metal-organic framework (MOF) material and an unwanted second material by disposing the solid mixture in a liquid separation medium having a density that lies between those of the wanted MOF material and the unwanted material, whereby the solid mixture separates by density differences into a fraction of wanted MOF material and another fraction of unwanted material.

  8. Slurried solid media for simultaneous water purification and carbon dioxide removal from gas mixtures

    DOEpatents

    Aines, Roger D.; Bourcier, William L.; Viani, Brian

    2013-01-29

    A slurried solid media for simultaneous water purification and carbon dioxide removal from gas mixtures includes the steps of dissolving the gas mixture and carbon dioxide in water providing a gas, carbon dioxide, water mixture; adding a porous solid media to the gas, carbon dioxide, water mixture forming a slurry of gas, carbon dioxide, water, and porous solid media; heating the slurry of gas, carbon dioxide, water, and porous solid media producing steam; and cooling the steam to produce purified water and carbon dioxide.

  9. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, H.; Smith, L.M.

    1997-01-07

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support. 3 figs.

  10. Extraction and Purification of R-phycoerythrin from Marine Red Algae.

    PubMed

    Dumay, Justine; Morançais, Michèle; Nguyen, Huu Phuo Trang; Fleurence, Joël

    2015-01-01

    This chapter focuses on the recovery of an R-Phycoerythrin (R-PE)-enriched fraction from marine algae. Since R-PE is a proteinaceous pigment, we have developed a simple and rapid two-step method devoted to the extraction and purification of R-PE from marine red algae. Here we describe a phosphate buffer extraction followed by anion exchange chromatography carried on a DEAE Sepharose Fast Flow column. To ensure the quality and quantity of R-PE recovery, we also indicate different methods to monitor each fraction obtained, such as spectrophotometric indicators, gel filtration, and SDS-PAGE analysis. PMID:26108500

  11. Expression and Purification of Recombinant CDKs: CDK7, CDK8, and CDK9.

    PubMed

    Pinhero, Reena; Yankulov, Krassimir

    2016-01-01

    Cyclin-dependent kinases have established roles in the regulation of cell cycle, in gene expression and in cell differentiation. Many of these kinases have been considered as drug targets and numerous efforts have been made to develop specific and potent inhibitors against them. The first step in all of these attempts and in many other biochemical analyses is the production of highly purified and reliable kinase, most frequently in a recombinant form. In this chapter we describe our experience in the cloning, expression, and purification of CDKs using CDK7/CycH, CDK8/CycC, and CDK9/CycT1 as an example. PMID:26231705

  12. Rapid purification of circular DNA by triplex-mediated affinity capture

    DOEpatents

    Ji, Huamin; Smith, Lloyd M.

    1997-01-01

    A single-step capture of a target supercoiled double-stranded DNA molecule is accomplished by forming a local triple-helix among two strands of the supercoiled circular DNA and an oligonucleotide probe. The oligonucleotide is bound to an immobilizing support which facilitates the immobilization and purification of target DNA molecules. Non-target DNA molecules and other contaminating cellular material are easily removed by washing. The triple-helical structure is destabilized by raising the pH, leaving purified target DNA in the supernatant and reusable affinity capture oligonucleotide secured to the immobilizing support.

  13. Experimental method for the purification and reconditioning of ferrofluids

    NASA Astrophysics Data System (ADS)

    Cotae, Constantin

    1987-03-01

    The paper presents the theoretical aspects regarding the magnetogravimetric purification of ferrofluids both in the process of preparation and for their reconditioning from impurities. An experimental device used for magnetogravimetric purification is described together with experiments on some samples of oil-based ferrofluid that became impure with non-mixible solid, liquid, magnetic and nonmagnetic ingredients. The experiments resulted in a complete purification of the ferrofluid samples.

  14. Solvent-extraction purification of neptunium

    SciTech Connect

    Kyser, E.A.; Hudlow, S.L.

    2008-07-01

    The Savannah River Site (SRS) has recovered {sup 237}Np from reactor fuel that is currently being processed into NpO{sub 2} for future production of {sup 238}Pu. Several purification flowsheets have been utilized. An oxidizing solvent-extraction (SX) flowsheet was used to remove Fe, sulfate ion, and Th while simultaneously {sup 237}Np, {sup 238}Pu, u, and nonradioactive Ce(IV) was extracted into the tributyl phosphate (TBP) based organic solvent. A reducing SX flowsheet (second pass) removed the Ce and Pu and recovered both Np and U. The oxidizing flowsheet was necessary for solutions that contained excessive amounts of sulfate ion. Anion exchange was used to perform final purification of Np from Pu, U, and various non-actinide impurities. The Np(IV) in the purified solution was then oxalate-precipitated and calcined to an oxide for shipment to other facilities for storage and future target fabrication. Performance details of the SX purification and process difficulties are discussed. (authors)

  15. Rotating Reverse-Osmosis for Water Purification

    NASA Technical Reports Server (NTRS)

    Lueptow, RIchard M.

    2004-01-01

    A new design for a water-filtering device combines rotating filtration with reverse osmosis to create a rotating reverse- osmosis system. Rotating filtration has been used for separating plasma from whole blood, while reverse osmosis has been used in purification of water and in some chemical processes. Reverse- osmosis membranes are vulnerable to concentration polarization a type of fouling in which the chemicals meant not to pass through the reverse-osmosis membranes accumulate very near the surfaces of the membranes. The combination of rotating filtration and reverse osmosis is intended to prevent concentration polarization and thereby increase the desired flux of filtered water while decreasing the likelihood of passage of undesired chemical species through the filter. Devices based on this concept could be useful in a variety of commercial applications, including purification and desalination of drinking water, purification of pharmaceutical process water, treatment of household and industrial wastewater, and treatment of industrial process water. A rotating filter consists of a cylindrical porous microfilter rotating within a stationary concentric cylindrical outer shell (see figure). The aqueous suspension enters one end of the annulus between the inner and outer cylinders. Filtrate passes through the rotating cylindrical microfilter and is removed via a hollow shaft. The concentrated suspension is removed at the end of the annulus opposite the end where the suspension entered.

  16. Overview of the Purification of Recombinant Proteins

    PubMed Central

    Wingfield, Paul T.

    2015-01-01

    When the first version of this unit was written in 1995 protein purification of recombinant proteins was based on a variety of standard chromatographic methods and approaches many of which were described and mentioned in this unit and elsewhere in the book. In the interim there has been a shift towards an almost universal usage of the affinity or fusion tag. This may not be the case for biotechnology manufacture where affinity tags can complicate producing proteins under regulatory conditions. Regardless of the protein expression system, questions are asked as to which and how many affinity tags to use, where to attach them in the protein and whether to engineer a self cleavage system or simply leave them on. We will briefly address some of these issues. Also although this overview focuses on E.coli, protein expression and purification from the other commonly used expression systems are mentioned and apart from cell breakage methods, the protein purification methods and strategies are essentially the same. PMID:25829302

  17. Experimental purification of two-atom entanglement

    NASA Astrophysics Data System (ADS)

    Reichle, R.; Leibfried, D.; Knill, E.; Britton, J.; Blakestad, R. B.; Jost, J. D.; Langer, C.; Ozeri, R.; Seidelin, S.; Wineland, D. J.

    2006-10-01

    Entanglement is a necessary resource for quantum applications-entanglement established between quantum systems at different locations enables private communication and quantum teleportation, and facilitates quantum information processing. Distributed entanglement is established by preparing an entangled pair of quantum particles in one location, and transporting one member of the pair to another location. However, decoherence during transport reduces the quality (fidelity) of the entanglement. A protocol to achieve entanglement `purification' has been proposed to improve the fidelity after transport. This protocol uses separate quantum operations at each location and classical communication to distil high-fidelity entangled pairs from lower-fidelity pairs. Proof-of-principle experiments distilling entangled photon pairs have been carried out. However, these experiments obtained distilled pairs with a low probability of success and required destruction of the entangled pairs, rendering them unavailable for further processing. Here we report efficient and non-destructive entanglement purification with atomic quantum bits. Two noisy entangled pairs were created and distilled into one higher-fidelity pair available for further use. Success probabilities were above 35 per cent. The many applications of entanglement purification make it one of the most important techniques in quantum information processing.

  18. Experimental purification of two-atom entanglement.

    PubMed

    Reichle, R; Leibfried, D; Knill, E; Britton, J; Blakestad, R B; Jost, J D; Langer, C; Ozeri, R; Seidelin, S; Wineland, D J

    2006-10-19

    Entanglement is a necessary resource for quantum applications--entanglement established between quantum systems at different locations enables private communication and quantum teleportation, and facilitates quantum information processing. Distributed entanglement is established by preparing an entangled pair of quantum particles in one location, and transporting one member of the pair to another location. However, decoherence during transport reduces the quality (fidelity) of the entanglement. A protocol to achieve entanglement 'purification' has been proposed to improve the fidelity after transport. This protocol uses separate quantum operations at each location and classical communication to distil high-fidelity entangled pairs from lower-fidelity pairs. Proof-of-principle experiments distilling entangled photon pairs have been carried out. However, these experiments obtained distilled pairs with a low probability of success and required destruction of the entangled pairs, rendering them unavailable for further processing. Here we report efficient and non-destructive entanglement purification with atomic quantum bits. Two noisy entangled pairs were created and distilled into one higher-fidelity pair available for further use. Success probabilities were above 35 per cent. The many applications of entanglement purification make it one of the most important techniques in quantum information processing. PMID:17051214

  19. Purification of FGD gypsum product

    SciTech Connect

    Rogers, K.J.; Owens, F.C. II.

    1993-06-01

    A method of purifying a gypsum slurry resulting from an upstream flue gas desulfurization process is described comprising the steps of: (a) delivering the gypsum slurry to a primary dewatering device; (b) separating this gypsum slurry in said primary dewatering device into a coarse solids stream and a fine solids stream, said coarse solids stream primarily containing coarse particle sizes therein and said fine solids stream primarily containing fine particle sizes therein; (c) selectively returning all or a portion of said fine solids stream back to the upstream flue gas desulfurization process or delivering all or a portion of said fine solids stream to downstream separation means for further separation into a thickened fines stream and a process water stream, said process water stream thereafter being selectively delivered, as desired, to the upstream flue gas desulfurization process; (d) delivering said coarse solids stream to a surge/mix tank where it is selectively mixed with a portion of said thickened fines stream prior to being delivered to a secondary dewatering device; and, (e) collecting a purified gypsum product from said secondary dewatering device.

  20. Nucleation of crystals from solution: classical and two-step models.

    PubMed

    Erdemir, Deniz; Lee, Alfred Y; Myerson, Allan S

    2009-05-19

    Crystallization is vital to many processes occurring in nature and in the chemical, pharmaceutical, and food industries. Notably, crystallization is an attractive isolation step for manufacturing because this single process combines both particle formation and purification. Almost all of the products based on fine chemicals, such as dyes, explosives, and photographic materials, require crystallization in their manufacture, and more than 90% of all pharmaceutical products contain bioactive drug substances and excipients in the crystalline solid state. Hence control over the crystallization process allows manufacturers to obtain products with desired and reproducible properties. We judge the quality of a crystalline product based on four main properties: size, purity, morphology, and crystal structure. The pharmaceutical industry in particular requires production of the desired crystal form (polymorph) to assure the bioavailability and stability of the drug substance. In solution crystallization, nucleation plays a decisive role in determining the crystal structure and size distribution. Therefore, understanding the fundamentals of nucleation is crucial to achieve control over these properties. Because of its analytical simplicity, researchers have widely applied classical nucleation theory to solution crystallization. However, a number of differences between theoretical predictions and experimental results suggest that nucleation of solids from solution does not proceed via the classical pathway but follows more complex routes. In this Account, we discuss the shortcomings of classical nucleation theory and review studies contributing to the development of the modern two-step model. In the two-step model that was initially proposed for protein crystallization, a sufficient-sized cluster of solute molecules forms first, followed by reorganization of that cluster into an ordered structure. In recent experimental and theoretical studies, we and other researchers have