An effective purification method using large bottles for human pancreatic islet isolation

PubMed Central

Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A.; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F.; Grayburn, Paul A.; Matsumoto, Shinichi

2012-01-01

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification. PMID:23221740

Necessity of purification during bacterial DNA extraction with environmental soils

PubMed Central

Choi, Jung-Hyun

2017-01-01

Complexity and heterogeneity of soil samples have often implied the inclusion of purification steps in conventional DNA extraction for polymerase chain reaction (PCR) assays. Unfortunately the purification steps are also time and labor intensive. Therefore the necessity of DNA purification was re-visited and investigated for a variety of environmental soil samples that contained various amounts of PCR inhibitors. Bead beating and centrifugation was used as the baseline (without purification) method for DNA extraction. Its performance was compared with that of conventional DNA extraction kit (with purification). The necessity criteria for DNA purification were established with environmental soil samples. Using lysis conditions at 3000 rpm for 3 minutes with 0.1 mm glass beads, centrifugation time of 10 minutes and 1:10 dilution ratio, the baseline method outperformed conventional DNA extraction on cell seeded sand samples. Further investigation with PCR inhibitors (i.e., humic acids, clay, and magnesium [Mg]) showed that sand samples containing less than 10 μg/g humic acids and 70% clay may not require purifications. Interestingly, the inhibition pattern of Mg ion was different from other inhibitors due to the complexation interaction of Mg ion with DNA fragments. It was concluded that DNA extraction method without purification is suitable for soil samples that have less than 10 μg/g of humic acids, less than 70% clay content and less than 0.01% Mg ion content. PMID:28793754

Inert gas enhanced laser-assisted purification of platinum electron-beam-induced deposits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon

Electron-beam-induced deposition patterns, with composition of PtC 5, were purified using a pulsed laser-induced purification reaction to erode the amorphous carbon matrix and form pure platinum deposits. Enhanced mobility of residual H 2O molecules via a localized injection of inert Ar–H 2 (4%) is attributed to be the reactive gas species for purification of the deposits. Surface purification of deposits was realized at laser exposure times as low as 0.1 s. The ex situ purification reaction in the deposit interior was shown to be rate-limited by reactive gas diffusion into the deposit, and deposit contraction associated with the purification processmore » caused some loss of shape retention. To circumvent the intrinsic flaws of the ex situ anneal process, in situ deposition and purification techniques were explored that resemble a direct write atomic layer deposition (ALD) process. First, we explored a laser-assisted electron-beam-induced deposition (LAEBID) process augmented with reactive gas that resulted in a 75% carbon reduction compared to standard EBID. Lastly, a sequential deposition plus purification process was also developed and resulted in deposition of pure platinum deposits with high fidelity and shape retention.« less

Purification of crude glycerol from transesterification reaction of palm oil using direct method and multistep method

NASA Astrophysics Data System (ADS)

Nasir, N. F.; Mirus, M. F.; Ismail, M.

2017-09-01

Crude glycerol which produced from transesterification reaction has limited usage if it does not undergo purification process. It also contains excess methanol, catalyst and soap. Conventionally, purification method of the crude glycerol involves high cost and complex processes. This study aimed to determine the effects of using different purification methods which are direct method (comprises of ion exchange and methanol removal steps) and multistep method (comprises of neutralization, filtration, ion exchange and methanol removal steps). Two crude glycerol samples were investigated; the self-produced sample through the transesterification process of palm oil and the sample obtained from biodiesel plant. Samples were analysed using Fourier Transform Infrared Spectroscopy, Gas Chromatography and High Performance Liquid Chromatography. The results of this study for both samples after purification have showed that the pure glycerol was successfully produced and fatty acid salts were eliminated. Also, the results indicated the absence of methanol in both samples after purification process. In short, the combination of 4 purification steps has contributed to a higher quality of glycerol. Multistep purification method gave a better result compared to the direct method as neutralization and filtration steps helped in removing most excess salt, fatty acid and catalyst.

Inert gas enhanced laser-assisted purification of platinum electron-beam-induced deposits

DOE PAGES

Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon; ...

2015-06-30

Electron-beam-induced deposition patterns, with composition of PtC 5, were purified using a pulsed laser-induced purification reaction to erode the amorphous carbon matrix and form pure platinum deposits. Enhanced mobility of residual H 2O molecules via a localized injection of inert Ar–H 2 (4%) is attributed to be the reactive gas species for purification of the deposits. Surface purification of deposits was realized at laser exposure times as low as 0.1 s. The ex situ purification reaction in the deposit interior was shown to be rate-limited by reactive gas diffusion into the deposit, and deposit contraction associated with the purification processmore » caused some loss of shape retention. To circumvent the intrinsic flaws of the ex situ anneal process, in situ deposition and purification techniques were explored that resemble a direct write atomic layer deposition (ALD) process. First, we explored a laser-assisted electron-beam-induced deposition (LAEBID) process augmented with reactive gas that resulted in a 75% carbon reduction compared to standard EBID. Lastly, a sequential deposition plus purification process was also developed and resulted in deposition of pure platinum deposits with high fidelity and shape retention.« less

Affinity chromatography: A versatile technique for antibody purification.

PubMed

Arora, Sushrut; Saxena, Vikas; Ayyar, B Vijayalakshmi

2017-03-01

Antibodies continue to be extremely utilized entities in myriad applications including basic research, imaging, targeted delivery, chromatography, diagnostics, and therapeutics. At production stage, antibodies are generally present in complex matrices and most of their intended applications necessitate purification. Antibody purification has always been a major bottleneck in downstream processing of antibodies, due to the need of high quality products and associated high costs. Over the years, extensive research has focused on finding better purification methodologies to overcome this holdup. Among a plethora of different techniques, affinity chromatography is one of the most selective, rapid and easy method for antibody purification. This review aims to provide a detailed overview on affinity chromatography and the components involved in purification. An array of support matrices along with various classes of affinity ligands detailing their underlying working principles, together with the advantages and limitations of each system in purifying different types of antibodies, accompanying recent developments and important practical methodological considerations to optimize purification procedure are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Superparamagnetic poly(methyl methacrylate) beads for nattokinase purification from fermentation broth.

PubMed

Yang, Chengli; Xing, Jianmin; Guan, Yueping; Liu, Huizhou

2006-09-01

An effective method for purification of nattokinase from fermentation broth using magnetic poly(methyl methacrylate) (PMMA) beads immobilized with p-aminobenzamidine was proposed in this study. Firstly, magnetic PMMA beads with a narrow size distribution were prepared by spraying suspension polymerization. Then, they were highly functionalized via transesterification reaction with polyethylene glycol. The surface hydroxyl-modified magnetic beads obtained were further modified with chloroethylamine to transfer the surface amino-modified magnetic functional beads. The morphology and surface functionality of the magnetic beads were examined by scanning electron microscopy and Fourier transform infrared. An affinity ligand, p-aminobenzamidine was covalently immobilized to the amino-modified magnetic beads by the glutaraldehyde method for nattokinase purification directly from the fermentation broth. The purification factor and the recovery of the enzyme activity were found to be 8.7 and 85%, respectively. The purification of nattokinase from fermentation broth by magnetic beads only took 40 min, which shows a very fast purification of nattokinase compared to traditional purification methods.

The Blood Compatibilities of Blood Purification Membranes and Other Materials Developed in Japan

PubMed Central

Abe, Takaya; Kato, Karen; Fujioka, Tomoaki; Akizawa, Tadao

2011-01-01

The biocompatibilities in blood purification therapy are defined as “a concept to stipulate safety of blood purification therapy by an index based on interaction in the body arising from blood purification therapy itself.” The biocompatibilities are associated with not only materials to be used but also many factors such as sterilization method and eluted substance. It is often evaluated based on impacts on cellular pathways and on humoral pathways. Since the biocompatibilities of blood purification therapy in particular hemodialysis are not just a prognostic factor for dialysis patients but a contributory factor for long-term complications, it should be considered with adequate attention. It is important that blood purification therapy should be performed by consistently evaluating not only risks associated with these biocompatibilities but also the other advantages obtained from treatments. In this paper, the biocompatibilities of membrane and adsorption material based on Japanese original which are used for blood purification therapy are described. PMID:21969830

Evaluation of short purification cycles in naturally contaminated Mediterranean mussels (Mytilus galloprovincialis) harvested in Sardinia (Italy).

PubMed

Sferlazzo, Giovanni; Meloni, Domenico; Lamon, Sonia; Marceddu, Marta; Mureddu, Anna; Consolati, Simonetta Gianna; Pisanu, Margherita; Virgilio, Sebastiano

2018-09-01

The aim of the present study was to investigate the effect of short purification cycles on the safety of naturally contaminated Mytilus galloprovincialis from harvesting areas of the Gulf of Olbia (Sardinia, Italy). Samples from ten batches of mussels were collected before, during and after purification treatment at two purification centres (A-B). All the samples were analysed for Escherichia coli and Salmonella spp according to Council Regulation (EC) 2285/2015. Detection and enumeration of Vibrio spp were performed according to previously published methods. Presumptive identification of Vibrio spp isolates were performed by means of conventional biochemical tests and polymerase chain reaction. The presence of Hepatitis A virus was detected by nested reverse transcriptase-polymerase chain reaction. Environmental parameters (water temperature and salinity) were also recorded. The results of Escherichia coli counts showed the overall efficacy of the short purification cycles; a purification cycle of 8 h led to a rapid decline in the concentration. The decrease in Escherichia coli counts does not correlate with the presence of naturally occurring vibrios, the decline of which occurs at an even slower rate. The average contamination levels for Vibrio spp before purification were 8.20 ± 0.47 and 7.99 ± 0.62 Log 10  CFU/g in samples collected at purification plants A and B, respectively. After purification, the average contamination levels were 8.10 ± 0.60 Log 10  CFU/g at purification plant A and 7.85 ± 0.57 Log 10  CFU/g at purification plant B. The contaminated samples revealed the presence of Vibrio alginolyticus (n=21), Vibrio fluvialis (n=12), Vibrio cholerae (n=4), Vibrio parahaemolyticus (n=2) and Vibrio vulnificus (n=1). The Vibrio parahaemolyticus isolates carried the tdh or the trh genes. None of the isolates was tdh+/trh+. Salmonella spp and Hepatitis A virus were not detected. The adoption of short purification cycles for Mytilus galloprovincialis in the presence of pathogenic vibrios might not be sufficient to guarantee the safety of consumers. Copyright © 2018 Elsevier Ltd. All rights reserved.

Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns.

PubMed

Liu, Keyang; Wen, Zhanbo; Li, Na; Yang, Wenhui; Hu, Lingfei; Wang, Jie; Yin, Zhe; Dong, Xiaokai; Li, Jinsong

2012-12-01

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10-30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column. Copyright © 2012 Elsevier B.V. All rights reserved.

Evaluation of the effectiveness of semen storage and sperm purification methods for spermatozoa transcript profiling.

PubMed

Mao, Shihong; Goodrich, Robert J; Hauser, Russ; Schrader, Steven M; Chen, Zhen; Krawetz, Stephen A

2013-10-01

Different semen storage and sperm purification methods may affect the integrity of isolated spermatozoal RNA. RNA-Seq was applied to determine whether semen storage methods (pelleted vs. liquefied) and somatic cell lysis buffer (SCLB) vs. PureSperm (PS) purification methods affect the quantity and quality of sperm RNA. The results indicate that the method of semen storage does not markedly impact RNA profiling whereas the choice of purification can yield significant differences. RNA-Seq showed that the majority of mitochondrial and mid-piece associated transcripts were lost after SCLB purification, which indicated that the mid-piece of spermatozoa may have been compromised. In addition, the number of stable transcript pairs from SCLB-samples was less than that from the PS samples. This study supports the view that PS purification better maintains the integrity of spermatozoal RNAs.

Intensification of oily waste waters purification by means of liquid atomization

NASA Astrophysics Data System (ADS)

Eskin, A. A.; Tkach, N. S.; Kim, M. I.; Zakharov, G. A.

2017-10-01

In this research, a possibility of using liquid atomization for improving the efficiency of purification of wastewater by different methods has been studied. By the introduced method and an experimental setup for wastewater purification, saturation rate increases with its purification by means of dissolved air flotation. Liquid atomization under excess pressure allows to gain a large interfacial area between the saturated liquid and air, which may increase the rate of purified liquid saturation almost twice, compared to the existing methods of saturation. Current disadvantages of liquid atomization used for intensification of wastewater purification include high energy cost and secondary emulsion of polluting agents. It is also known that by means of liquid atomization a process of ozonizing can be intensified. Large contact surface between the purified liquid and ozone-air mixture increases the oxidizing efficiency, which allows to diminish ozone discharge. Liquid atomization may be used for purification of wastewaters by ultraviolet radiation. Small drops of liquid will be proportionally treated by ultraviolet, which makes it possible to do purification even of turbid wastewaters. High-speed liquid motion will prevent the pollution of quartz tubes of ultraviolet lamps.

Purification of inulinases by changing the ionic strength of the medium and precipitation with alcohols.

PubMed

Golunski, Simone; Silva, Marceli F; Marques, Camila T; Rosseto, Vanusa; Kaizer, Rosilene R; Mossi, Altemir J; Rigo, Diane; Dallago, Rogério M; DI Luccio, Marco; Treichel, Helen

2017-01-01

The present study evaluated the purification of inulinase by changing the ionic strength of the medium by addition of NaCl and CaCl2 followed by precipitation with n-propyl alcohol or iso-propyl alcohol. The effects of the concentration of alcohols and the rate of addition of alcohols in the crude extract on the purification yield and purification factor were evaluated. Precipitation caused an activation of enzyme and allowed purification factors up to 2.4-fold for both alcohols. The purification factor was affected positively by the modification of the ionic strength of the medium to 0.5 mol.L-1 NaCl before precipitation with the alcohol (n-propyl or iso-propyl). A purification factor of 4.8-fold and an enzyme yield of 78.1 % could be achieved by the addition of 0.5 mol.L-1 of NaCl to the crude extract, followed by the precipitation with 50 % (v/v) of n-propyl alcohol, added at a flow rate of 19.9 mL/min.

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.

Impulse powerful UV-radiation source pumped by the sublight ionization waves for the bacteriological disinfection of water

NASA Astrophysics Data System (ADS)

Filiouguine, Igor V.; Kostiouchenko, S. V.; Koudryavtsev, N. N.; Vasilyak, Leonid M.; Yakimenko, A. V.

1993-11-01

The bacteriological disinfective action of UV-radiation is well known. The pioneer work on UV-radiation used for bacteriological disinfection of waste water was made in 1910. Because of the high cost and low living time of the UV-radiation sources, the alternative technique for waste water purification by chlorine introducing was spread out. During the second stage of the UV purification development, beginning in approximately 1970, the interest for bacteriological cleaning of water, increased again. Two reasons were responsible for this event: first, the significant improvement of technology and design of UV-bacteriological purificators, and second, recognition of the serious danger of chlorine compounds introduced into water under purification because of the toxicity of these compounds. Further investigations gave excellent results in the creation and industrial applications of UV- bacteriological purificators. Now we can see a rapid development of industrial technology in UV-purification of drinking and waste waters.

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.

A rapid Orthopoxvirus purification protocol suitable for high-containment laboratories.

PubMed

Hughes, Laura; Wilkins, Kimberly; Goldsmith, Cynthia S; Smith, Scott; Hudson, Paul; Patel, Nishi; Karem, Kevin; Damon, Inger; Li, Yu; Olson, Victoria A; Satheshkumar, P S

2017-05-01

Virus purification in a high-containment setting provides unique challenges due to barrier precautions and operational safety approaches that are not necessary in lower biosafety level (BSL) 2 environments. The need for high risk group pathogen diagnostic assay development, anti-viral research, pathogenesis and vaccine efficacy research necessitates work in BSL-3 and BSL-4 labs with infectious agents. When this work is performed in accordance with BSL-4 practices, modifications are often required in standard protocols. Classical virus purification techniques are difficult to execute in a BSL-3 or BSL-4 laboratory because of the work practices used in these environments. Orthopoxviruses are a family of viruses that, in some cases, requires work in a high-containment laboratory and due to size do not lend themselves to simpler purification methods. Current CDC purification techniques of orthopoxviruses uses 1,1,2-trichlorotrifluoroethane, commonly known as Genetron ® . Genetron ® is a chlorofluorocarbon (CFC) that has been shown to be detrimental to the ozone and has been phased out and the limited amount of product makes it no longer a feasible option for poxvirus purification purposes. Here we demonstrate a new Orthopoxvirus purification method that is suitable for high-containment laboratories and produces virus that is not only comparable to previous purification methods, but improves on purity and yield. Published by Elsevier B.V.

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)

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 cost-effective, rapid, and reliable avenue for the purification of recombinant proteins in heterologous hosts. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

Continuous Purification of Colloidal Quantum Dots in Large-Scale Using Porous Electrodes in Flow Channel.

PubMed

Lim, Hosub; Woo, Ju Young; Lee, Doh C; Lee, Jinkee; Jeong, Sohee; Kim, Duckjong

2017-02-27

Colloidal quantum dots (QDs) afford huge potential in numerous applications owing to their excellent optical and electronic properties. After the synthesis of QDs, separating QDs from unreacted impurities in large scale is one of the biggest issues to achieve scalable and high performance optoelectronic applications. Thus far, however, continuous purification method, which is essential for mass production, has rarely been reported. In this study, we developed a new continuous purification process that is suitable to the mass production of high-quality QDs. As-synthesized QDs are driven by electrophoresis in a flow channel and captured by porous electrodes and finally separated from the unreacted impurities. Nuclear magnetic resonance and ultraviolet/visible/near-infrared absorption spectroscopic data clearly showed that the impurities were efficiently removed from QDs with the purification yield, defined as the ratio of the mass of purified QDs to that of QDs in the crude solution, up to 87%. Also, we could successfully predict the purification yield depending on purification conditions with a simple theoretical model. The proposed large-scale purification process could be an important cornerstone for the mass production and industrial use of high-quality QDs.

Continuous Purification of Colloidal Quantum Dots in Large-Scale Using Porous Electrodes in Flow Channel

NASA Astrophysics Data System (ADS)

Lim, Hosub; Woo, Ju Young; Lee, Doh Chang; Lee, Jinkee; Jeong, Sohee; Kim, Duckjong

2017-11-01

Colloidal Quantum dots (QDs) afford huge potential in numerous applications owing to their excellent optical and electronic properties. After the synthesis of QDs, separating QDs from unreacted impurities in large scale is one of the biggest issues to achieve scalable and high performance optoelectronic applications. Thus far, however, continuous purification method, which is essential for mass production, has rarely been reported. In this study, we developed a new continuous purification process that is suitable to the mass production of high-quality QDs. As-synthesized QDs are driven by electrophoresis in a flow channel and captured by porous electrodes and finally separated from the unreacted impurities. Nuclear magnetic resonance and ultraviolet/visible/near-infrared absorption spectroscopic data clearly showed that the impurities were efficiently removed from QDs with the purification yield, defined as the ratio of the mass of purified QDs to that of QDs in the crude solution, up to 87%. Also, we could successfully predict the purification yield depending on purification conditions with a simple theoretical model. The proposed large-scale purification process could be an important cornerstone for the mass production and industrial use of high-quality QDs.

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.

A Family of LIC Vectors for High-Throughput Cloning and Purification of Proteins1

PubMed Central

Eschenfeldt, William H.; Stols, Lucy; Millard, Cynthia Sanville; Joachimiak, Andrzej; Donnelly, Mark I.

2009-01-01

Summary Fifteen related ligation-independent cloning vectors were constructed for high-throughput cloning and purification of proteins. The vectors encode a TEV protease site for removal of tags that facilitate protein purification (his-tag) or improve solubility (MBP, GST). Specialized vectors allow coexpression and copurification of interacting proteins, or in vivo removal of MBP by TVMV protease to improve screening and purification. All target genes and vectors are processed by the same protocols, which we describe here. PMID:18988021

Virus purification by CsCl density gradient using general centrifugation.

PubMed

Nasukawa, Tadahiro; Uchiyama, Jumpei; Taharaguchi, Satoshi; Ota, Sumire; Ujihara, Takako; Matsuzaki, Shigenobu; Murakami, Hironobu; Mizukami, Keijirou; Sakaguchi, Masahiro

2017-11-01

Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g, 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g, 1 h, 4 °C) using phages S13' and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

PubMed

Dong, Dexian; Gui, Yanli; Chen, Dezhao; Li, Rongxiu

2008-01-01

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. 2008 John Wiley & Sons, Ltd

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.

Photocatalytic materials and technologies for air purification.

PubMed

Ren, Hangjuan; Koshy, Pramod; Chen, Wen-Fan; Qi, Shaohua; Sorrell, Charles Christopher

2017-03-05

Since there is increasing concern for the impact of air quality on human health, the present work surveys the materials and technologies for air purification using photocatalytic materials. The coverage includes (1) current photocatalytic materials for the decomposition of chemical contaminants and disinfection of pathogens present in air and (2) photocatalytic air purification systems that are used currently and under development. The present work focuses on five main themes. First, the mechanisms of photodegradation and photodisinfection are explained. Second, system designs for photocatalytic air purification are surveyed. Third, the photocatalytic materials used for air purification and their characteristics are considered, including both conventional and more recently developed photocatalysts. Fourth, the methods used to fabricate these materials are discussed. Fifth, the most significant coverage is devoted to materials design strategies aimed at improving the performance of photocatalysts for air purification. The review concludes with a brief consideration of promising future directions for materials research in photocatalysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Retrospective analyses of the bottleneck in purification of eukaryotic proteins from Escherichia coli as affected by molecular weight, cysteine content and isoelectric point

PubMed Central

Jeon, Won Bae

2015-01-01

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value. PMID:20510014

Experimental Optimal Single Qubit Purification in an NMR Quantum Information Processor

PubMed Central

Hou, Shi-Yao; Sheng, Yu-Bo; Feng, Guan-Ru; Long, Gui-Lu

2014-01-01

High quality single qubits are the building blocks in quantum information processing. But they are vulnerable to environmental noise. To overcome noise, purification techniques, which generate qubits with higher purities from qubits with lower purities, have been proposed. Purifications have attracted much interest and been widely studied. However, the full experimental demonstration of an optimal single qubit purification protocol proposed by Cirac, Ekert and Macchiavello [Phys. Rev. Lett. 82, 4344 (1999), the CEM protocol] more than one and half decades ago, still remains an experimental challenge, as it requires more complicated networks and a higher level of precision controls. In this work, we design an experiment scheme that realizes the CEM protocol with explicit symmetrization of the wave functions. The purification scheme was successfully implemented in a nuclear magnetic resonance quantum information processor. The experiment fully demonstrated the purification protocol, and showed that it is an effective way of protecting qubits against errors and decoherence. PMID:25358758

Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425

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.

Single-step affinity purification for fungal proteomics.

PubMed

Liu, Hui-Lin; Osmani, Aysha H; Ukil, Leena; Son, Sunghun; Markossian, Sarine; Shen, Kuo-Fang; Govindaraghavan, Meera; Varadaraj, Archana; Hashmi, Shahr B; De Souza, Colin P; Osmani, Stephen A

2010-05-01

A single-step protein affinity purification protocol using Aspergillus nidulans is described. Detailed protocols for cell breakage, affinity purification, and depending on the application, methods for protein release from affinity beads are provided. Examples defining the utility of the approaches, which should be widely applicable, are included.

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.

Using an FPLC to Promote Active Learning of the Principles of Protein Structure and Purification

ERIC Educational Resources Information Center

Robinson, Rebekah L.; Neely, Amy E.; Mojadedi, Wais; Threatt, Katie N.; Davis, Nicole Y.; Weiland, Mitch H.

2017-01-01

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory…

Item Purification in Differential Item Functioning Using Generalized Linear Mixed Models

ERIC Educational Resources Information Center

Liu, Qian

2011-01-01

For this dissertation, four item purification procedures were implemented onto the generalized linear mixed model for differential item functioning (DIF) analysis, and the performance of these item purification procedures was investigated through a series of simulations. Among the four procedures, forward and generalized linear mixed model (GLMM)…

An Adaptable Investigative Graduate Laboratory Course for Teaching Protein Purification

ERIC Educational Resources Information Center

Carroll, Christopher W.; Keller, Lani C.

2014-01-01

This adaptable graduate laboratory course on protein purification offers students the opportunity to explore a wide range of techniques while allowing the instructor the freedom to incorporate their own personal research interests. The course design involves two sequential purification schemes performed in a single semester. The first part…

[Progress in isolation and purification of porcine islets].

PubMed

Zhu, Haitao; Yu, Liang; Wang, Bo

2012-08-01

To review the common methods of isolation and purification of porcine islets and research progress. Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.

Method for the purification of noble gases, nitrogen and hydrogen

DOEpatents

Baker, J.D.; Meikrantz, D.H.; Tuggle, D.G.

1997-09-23

A method and apparatus are disclosed for the purification and collection of hydrogen isotopes in a flowing inert gaseous mixture containing impurities, wherein metal alloy getters having the capability of sorbing non-hydrogen impurities such as oxygen, carbon dioxide, carbon monoxide, methane, ammonia, nitrogen and water vapor are utilized to purify the gaseous mixture of impurities. After purification hydrogen isotopes may be more efficiently collected. A plurality of parallel process lines utilizing metal getter alloys can be used to provide for the continuous purification and collection of the hydrogen isotopes. 15 figs.

Method for the purification of noble gases, nitrogen and hydrogen

DOEpatents

Baker, John D.; Meikrantz, David H.; Tuggle, Dale G.

1997-01-01

A method and apparatus for the purification and collection of hydrogen isotopes in a flowing inert gaseous mixture containing impurities, wherein metal alloy getters having the capability of sorbing non-hydrogen impurities such as oxygen, carbon dioxide, carbon monoxide, methane, ammonia, nitrogen and water vapor are utilized to purify the gaseous mixture of impurities. After purification hydrogen isotopes may be more efficiently collected. A plurality of parallel process lines utilizing metal getter alloys can be used to provide for the continuous purification and collection of the hydrogen isotopes.

Renaissance of protein crystallization and precipitation in biopharmaceuticals purification.

PubMed

Dos Santos, Raquel; Carvalho, Ana Luísa; Roque, A Cecília A

The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification of white spot syndrome virus by iodixanol density gradient centrifugation.

PubMed

Dantas-Lima, J J; Corteel, M; Cornelissen, M; Bossier, P; Sorgeloos, P; Nauwynck, H J

2013-10-01

Up to now, only a few brief procedures for purifying white spot syndrome virus (WSSV) have been described. They were mainly based on sucrose, NaBr and CsCl density gradient centrifugation. This work describes for the first time the purification of WSSV through iodixanol density gradients, using virus isolated from infected tissues and haemolymph of Penaeus vannamei (Boone). The purification from tissues included a concentration step by centrifugation (2.5 h at 60,000 g) onto a 50% iodixanol cushion and a purification step by centrifugation (3 h at 80,000 g) through a discontinuous iodixanol gradient (phosphate-buffered saline, 5%, 10%, 15% and 20%). The purification from infected haemolymph enclosed a dialysis step with a membrane of 1,000 kDa (18 h) and a purification step through the earlier iodixanol gradient. The gradients were collected in fractions and analysed. The number of particles, infectivity titre (in vivo), total protein and viral protein content were evaluated. The purification from infected tissues gave WSSV suspensions with a very high infectivity and an acceptable purity, while virus purified from haemolymph had a high infectivity and a very high purity. Additionally, it was observed that WSSV has an unusually low buoyant density and that it is very sensitive to high external pressures. © 2013 John Wiley & Sons Ltd.

Performance of photocatalyst based carbon nanodots from waste frying oil in water purification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aji, Mahardika Prasetya, E-mail: mahardika190@gmail.com; Wiguna, Pradita Ajeng; Susanto,

Carbon Nanodots (C-Dots) from waste frying oil could be used as a photocatalyst in water purification with solar light irradiation. Performance of C-Dots as a photocatalyst was tested in the process of water purification with a given synthetic sewage methylene blue. The tested was also conducted by comparing the performance C-Dots made from frying oil, waste fryng oil as a photocatalyst and solution of methylene blue without photocatalyst C-Dots. Performance of C-Dots from waste frying oil were estimated by the results of absorbance spectrum. The results of measurement absorbance spectrum from the process of water purification with photocatalyst C-Dots showedmore » that the highest intensity at a wavelength 664 nm of methylene blue decreased. The test results showed that the performance of photocatalyst C-Dots from waste frying oil was better in water purification. This estimated that number of particles C-dots is more in waste frying oil because have experieced repeated the heating process so that the higher particles concentration make the photocatalyst process more effective. The observation of the performance C-Dots from waste frying oil as a photocatalyst in the water purification processes become important invention for solving the problems of waste and water purification.« less

Purification of nanogram-range immunoprecipitated DNA in ChIP-seq application.

PubMed

Zhong, Jian; Ye, Zhenqing; Lenz, Samuel W; Clark, Chad R; Bharucha, Adil; Farrugia, Gianrico; Robertson, Keith D; Zhang, Zhiguo; Ordog, Tamas; Lee, Jeong-Heon

2017-12-21

Chromatin immunoprecipitation-sequencing (ChIP-seq) is a widely used epigenetic approach for investigating genome-wide protein-DNA interactions in cells and tissues. The approach has been relatively well established but several key steps still require further improvement. As a part of the procedure, immnoprecipitated DNA must undergo purification and library preparation for subsequent high-throughput sequencing. Current ChIP protocols typically yield nanogram quantities of immunoprecipitated DNA mainly depending on the target of interest and starting chromatin input amount. However, little information exists on the performance of reagents used for the purification of such minute amounts of immunoprecipitated DNA in ChIP elution buffer and their effects on ChIP-seq data. Here, we compared DNA recovery, library preparation efficiency, and ChIP-seq results obtained with several commercial DNA purification reagents applied to 1 ng ChIP DNA and also investigated the impact of conditions under which ChIP DNA is stored. We compared DNA recovery of ten commercial DNA purification reagents and phenol/chloroform extraction from 1 to 50 ng of immunopreciptated DNA in ChIP elution buffer. The recovery yield was significantly different with 1 ng of DNA while similar in higher DNA amounts. We also observed that the low nanogram range of purified DNA is prone to loss during storage depending on the type of polypropylene tube used. The immunoprecipitated DNA equivalent to 1 ng of purified DNA was subject to DNA purification and library preparation to evaluate the performance of four better performing purification reagents in ChIP-seq applications. Quantification of library DNAs indicated the selected purification kits have a negligible impact on the efficiency of library preparation. The resulting ChIP-seq data were comparable with the dataset generated by ENCODE consortium and were highly correlated between the data from different purification reagents. This study provides comparative data on commercial DNA purification reagents applied to nanogram-range immunopreciptated ChIP DNA and evidence for the importance of storage conditions of low nanogram-range purified DNA. We verified consistent high performance of a subset of the tested reagents. These results will facilitate the improvement of ChIP-seq methodology for low-input applications.

Tetanus toxoid purification: chromatographic procedures as an alternative to ammonium-sulphate precipitation.

PubMed

Stojićević, Ivana; Dimitrijević, Ljiljana; Dovezenski, Nebojša; Živković, Irena; Petrušić, Vladimir; Marinković, Emilija; Inić-Kanada, Aleksandra; Stojanović, Marijana

2011-08-01

Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin's biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. Copyright © 2011 Elsevier B.V. All rights reserved.

Purification of Proteins From Cell-Culture Medium or Cell-Lysate by High-Speed Counter-Current Chromatography Using Cross-Axis Coil Planet Centrifuge

PubMed Central

Shibusawa, Yoichi; Ito, Yoichiro

2014-01-01

This review describes protein purifications from cell culture medium or cell-lysate by high speed counter-current chromatography using the cross-axis coil planet centrifuge. Purifications were performed using aqueous two phase systems composed of polyethylene glycols and dextrans. PMID:25360182

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…

Purification of a Recombinant Glutathione Transferase from the Causative Agent of Hydatidosis, "Echinococcus granulosus"

ERIC Educational Resources Information Center

Fleitas, Andrea L.; Randall, Lía M.; Möller, Matías N.; Denicola, Ana

2016-01-01

This practical class activity was designed to introduce students to recombinant protein expression and purification. The principal goal is to shed light on basic aspects concerning recombinant protein production, in particular protein expression, chromatography methods for protein purification, and enzyme activity as a tool to evaluate purity and…

The MIMIC Method with Scale Purification for Detecting Differential Item Functioning

ERIC Educational Resources Information Center

Wang, Wen-Chung; Shih, Ching-Lin; Yang, Chih-Chien

2009-01-01

This study implements a scale purification procedure onto the standard MIMIC method for differential item functioning (DIF) detection and assesses its performance through a series of simulations. It is found that the MIMIC method with scale purification (denoted as M-SP) outperforms the standard MIMIC method (denoted as M-ST) in controlling…

Effect of Purification Procedures on DIF Analysis in IRTPRO

ERIC Educational Resources Information Center

Fikis, David R. J.; Oshima, T. C.

2017-01-01

Purification of the test has been a well-accepted procedure in enhancing the performance of tests for differential item functioning (DIF). As defined by Lord, purification requires reestimation of ability parameters after removing DIF items before conducting the final DIF analysis. IRTPRO 3 is a recently updated program for analyses in item…

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 SERVICES (CONTINUED) MEDICAL DEVICES GASTROENTEROLOGY-UROLOGY DEVICES Therapeutic Devices § 876.5665 Water purification system for hemodialysis. (a)...

A Versatile and Inexpensive Enzyme Purification Experiment for Undergraduate Biochemistry Labs.

ERIC Educational Resources Information Center

Farrell, Shawn O.; Choo, Darryl

1989-01-01

Develops an experiment that could be done in two- to three-hour blocks and does not rely on cold room procedures for most of the purification. Describes the materials, methods, and results of the purification of bovine heart lactate dehydrogenase using ammonium sulfate fractionation, dialysis, and separation using affinity chromatography and…

Evaluation of immobilized metal membrane affinity chromatography for purification of an immunoglobulin G1 monoclonal antibody.

PubMed

Serpa, Gisele; Augusto, Elisabeth Fátima Pires; Tamashiro, Wirla Maria Silva Cunha; Ribeiro, Mariana Borçoe; Miranda, Everson Alves; Bueno, Sônia Maria Alves

2005-02-25

The large scale production of monoclonal antibodies (McAbs) has gaining increased relevance with the development of the hybridoma cell culture in bioreactors creating a need for specific efficient bioseparation techniques. Conventional fixed bead affinity adsorption commonly applied for McAbs purification has the drawback of low flow rates and colmatage. We developed and evaluated a immobilized metal affinity chromatographies (IMAC) affinity membrane for the purification of anti-TNP IgG(1) mouse McAbs. We immobilized metal ions on a poly(ethylene vinyl alcohol) hollow fiber membrane (Me(2+)-IDA-PEVA) and applied it for the purification of this McAbs from cell culture supernatant after precipitation with 50% saturation of ammonium sulphate. The purity of IgG(1) in the eluate fractions was high when eluted from Zn(2+) complex. The anti-TNP antibody could be eluted under conditions causing no loss of antigen binding capacity. The purification procedure can be considered as an alternative to the biospecific adsorbent commonly applied for mouse IgG(1) purification, the protein G-Sepharose.

Development of the protocol for purification of artemisinin based on combination of commercial and computationally designed adsorbents.

PubMed

Piletska, Elena V; Karim, Kal; Cutler, Malcolm; Piletsky, Sergey A

2013-01-01

A polymeric adsorbent for extraction of the antimalarial drug artemisinin from Artemisia annua L. was computationally designed. This polymer demonstrated a high capacity for artemisinin (120 mg g(-1) ), quantitative recovery (87%) and was found to be an effective material for purification of artemisinin from complex plant matrix. The artemisinin quantification was conducted using an optimised HPLC-MS protocol, which was characterised by high precision and linearity in the concentration range between 0.05 and 2 μg mL(-1) . Optimisation of the purification protocol also involved screening of commercial adsorbents for the removal of waxes and other interfering natural compounds, which inhibit the crystallisation of artemisinin. As a result of a two step-purification protocol crystals of artemisinin were obtained, and artemisinin purity was evaluated as 75%. By performing the second stage of purification twice, the purity of artemisinin can be further improved to 99%. The developed protocol produced high-purity artemisinin using only a few purification steps that makes it suitable for large scale industrial manufacturing process. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Discussion on runoff purification technology of highway bridge deck based on water quality safety

NASA Astrophysics Data System (ADS)

Tan, Sheng-guang; Liu, Xue-xin; Zou, Guo-ping; Xiong, Xin-zhu; Tao, Shuang-cheng

2018-06-01

Aiming at the actual problems existing, including a poor purification effect of highway bridge runoff collection and treatment system across sensitive water and necessary manual emergency operation, three kinds of technology, three pools system of bridge runoff purification, the integral pool of bridge runoff purification and ecological planting tank, are put forward by optimizing the structure of purification unit and system setting. At the same time, we come up with an emergency strategy for hazardous material leakage basing on automatic identification and remote control of traffic accidents. On the basis of combining these with the optimized pool structure, sensitive water safety can be guaranteed and water pollution, from directly discharging of bridge runoff, can be decreased. For making up for the shortages of green highway construction technology, the technique has important reference value.

Improving the large scale purification of the HIV microbicide, griffithsin.

PubMed

Fuqua, Joshua L; Wanga, Valentine; Palmer, Kenneth E

2015-02-22

Griffithsin is a broad spectrum antiviral lectin that inhibits viral entry and maturation processes through binding clusters of oligomannose glycans on viral envelope glycoproteins. An efficient, scaleable manufacturing process for griffithsin active pharmaceutical ingredient (API) is essential for particularly cost-sensitive products such as griffithsin -based topical microbicides for HIV-1 prevention in resource poor settings. Our previously published purification method used ceramic filtration followed by two chromatography steps, resulting in a protein recovery of 30%. Our objective was to develop a scalable purification method for griffithsin expressed in Nicotiana benthamiana plants that would increase yield, reduce production costs, and simplify manufacturing techniques. Considering the future need to transfer griffithsin manufacturing technology to resource poor areas, we chose to focus modifying the purification process, paying particular attention to introducing simple, low-cost, and scalable procedures such as use of temperature, pH, ion concentration, and filtration to enhance product recovery. We achieved >99% pure griffithsin API by generating the initial green juice extract in pH 4 buffer, heating the extract to 55°C, incubating overnight with a bentonite MgCl2 mixture, and final purification with Capto™ multimodal chromatography. Griffithsin extracted with this protocol maintains activity comparable to griffithsin purified by the previously published method and we are able to recover a substantially higher yield: 88 ± 5% of griffithsin from the initial extract. The method was scaled to produce gram quantities of griffithsin with high yields, low endotoxin levels, and low purification costs maintained. The methodology developed to purify griffithsin introduces and develops multiple tools for purification of recombinant proteins from plants at an industrial scale. These tools allow for robust cost-effective production and purification of griffithsin. The methodology can be readily scaled to the bench top or industry and process components can be used for purification of additional proteins based on biophysical characteristics.

Two-step purification method of vitellogenin from three teleost fish species: rainbow trout (Oncorhynchus mykiss), gudgeon (Gobio gobio) and chub (Leuciscus cephalus).

PubMed

Brion, F; Rogerieux, F; Noury, P; Migeon, B; Flammarion, P; Thybaud, E; Porcher, J M

2000-01-14

A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.

Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

PubMed

Shi, Changhua; Han, Tzu-Chiang; Wood, David W

2017-01-01

Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean.

PubMed

Demeke, Tigst; Ratnayaka, Indira; Phan, Anh

2009-01-01

The quality of DNA affects the accuracy and repeatability of quantitative PCR results. Different DNA extraction and purification methods were compared for quantification of Roundup Ready (RR) soybean (event 40-3-2) by real-time PCR. DNA was extracted using cetylmethylammonium bromide (CTAB), DNeasy Plant Mini Kit, and Wizard Magnetic DNA purification system for food. CTAB-extracted DNA was also purified using the Zymo (DNA Clean & Concentrator 25 kit), Qtip 100 (Qiagen Genomic-Tip 100/G), and QIAEX II Gel Extraction Kit. The CTAB extraction method provided the largest amount of DNA, and the Zymo purification kit resulted in the highest percentage of DNA recovery. The Abs260/280 and Abs260/230 ratios were less than the expected values for some of the DNA extraction and purification methods used, indicating the presence of substances that could inhibit PCR reactions. Real-time quantitative PCR results were affected by the DNA extraction and purification methods used. Further purification or dilution of the CTAB DNA was required for successful quantification of RR soybean. Less variability of quantitative PCR results was observed among experiments and replications for DNA extracted and/or purified by CTAB, CTAB+Zymo, CTAB+Qtip 100, and DNeasy methods. Correct and repeatable results for real-time PCR quantification of RR soybean were achieved using CTAB DNA purified with Zymo and Qtip 100 methods.

Purification of liquid metal systems with sodium coolant from oxygen using getters

NASA Astrophysics Data System (ADS)

Kozlov, F. A.; Konovalov, M. A.; Sorokin, A. P.

2016-05-01

For increasing the safety and economic parameters of nuclear power stations (NPSs) with sodium coolant, it was decided to install all systems contacting radioactive sodium, including purification systems of circuit I, in the reactor vessel. The performance and capacity of cold traps (CTs) (conventional element of coolant purification systems) in these conditions are limited by their volume. It was proposed to use hot traps (HTs) in circuit I for coolant purification from oxygen. It was demonstrated that, at rated parameters of the installation when the temperature of the coolant streamlining the getter (gas absorber) is equal to 550°C, the hot trap can provide the required coolant purity. In shutdown modes at 250-300°C, the performance of the hot trap is reduced by four orders of magnitude. Possible HT operation regimes for shutdown modes and while reaching rated parameters were proposed and analyzed. Basic attention was paid to purification modes at power rise after commissioning and accidental contamination of the coolant when the initial oxygen concentration in it reached 25 mln-1. It was demonstrated that the efficiency of purification systems can be increased using HTs with the getter in the form of a foil or granules. The possibility of implementing the "fast purification" mode in which the coolant is purified simultaneously with passing over from the shutdown mode to the rated parameters was substantiated.

Method for Rapid Purification of Class IIa Bacteriocins and Comparison of Their Activities

PubMed Central

Guyonnet, D.; Fremaux, C.; Cenatiempo, Y.; Berjeaud, J. M.

2000-01-01

A three-step method was developed for the purification of mesentericin Y105 (60% yield) from the culture supernatant of Leuconostoc mesenteroides Y105. The same procedure was successfully applied to the purification of five other anti-Listeria bacteriocins identified by mass spectrometry. Specific activities of the purified bacteriocins were compared. PMID:10742275

Item Purification Does Not Always Improve DIF Detection: A Counterexample with Angoff's Delta Plot

ERIC Educational Resources Information Center

Magis, David; Facon, Bruno

2013-01-01

Item purification is an iterative process that is often advocated as improving the identification of items affected by differential item functioning (DIF). With test-score-based DIF detection methods, item purification iteratively removes the items currently flagged as DIF from the test scores to get purified sets of items, unaffected by DIF. The…

A novel method for purification of the endogenously expressed fission yeast Set2 complex.

PubMed

Suzuki, Shota; Nagao, Koji; Obuse, Chikashi; Murakami, Yota; Takahata, Shinya

2014-05-01

Chromatin-associated proteins are heterogeneously and dynamically composed. To gain a complete understanding of DNA packaging and basic nuclear functions, it is important to generate a comprehensive inventory of these proteins. However, biochemical purification of chromatin-associated proteins is difficult and is accompanied by concerns over complex stability, protein solubility and yield. Here, we describe a new method for optimized purification of the endogenously expressed fission yeast Set2 complex, histone H3K36 methyltransferase. Using the standard centrifugation procedure for purification, approximately half of the Set2 protein separated into the insoluble chromatin pellet fraction, making it impossible to recover the large amounts of soluble Set2. To overcome this poor recovery, we developed a novel protein purification technique termed the filtration/immunoaffinity purification/mass spectrometry (FIM) method, which eliminates the need for centrifugation. Using the FIM method, in which whole cell lysates were filtered consecutively through eight different pore sizes (53-0.8μm), a high yield of soluble FLAG-tagged Set2 was obtained from fission yeast. The technique was suitable for affinity purification and produced a low background. A mass spectrometry analysis of anti-FLAG immunoprecipitated proteins revealed that Rpb1, Rpb2 and Rpb3, which have all been reported previously as components of the budding yeast Set2 complex, were isolated from fission yeast using the FIM method. In addition, other subunits of RNA polymerase II and its phosphatase were also identified. In conclusion, the FIM method is valid for the efficient purification of protein complexes that separate into the insoluble chromatin pellet fraction during centrifugation. Copyright © 2014 Elsevier Inc. All rights reserved.

Experimental study and simulation of phosphorus purification effects of bioretention systems on urban surface runoff

PubMed Central

Liang, Zheng; Li, Yajiao; Li, Peng; Jiang, Chunbo

2018-01-01

Excessive phosphorus (P) contributes to eutrophication by degrading water quality and limiting human use of water resources. Identifying economic and convenient methods to control soluble reactive phosphorus (SRP) pollution in urban runoff is the key point of rainwater management strategies. Through three series of different tests involving influencing factors, continuous operation and intermittent operation, this study explored the purification effects of bioretention tanks under different experimental conditions, it included nine intermittent tests, single field continuous test with three groups of different fillers (Fly ash mixed with sand, Blast furnace slag, and Soil), and eight intermittent tests with single filler (Blast furnace slag mixed with sand). Among the three filler combinations studied, the filler with fly ash mixed with sand achieved the best pollution reduction efficiency. The setting of the submerged zone exerted minimal influence on the P removal of the three filler combinations. An extension of the dry period slightly promoted the P purification effect. The combination of fly ash mixed with sand demonstrated a positive purification effect on SRP during short- or long-term simulated rainfall duration. Blast furnace slag also presented a positive purification effect in the short term, although its continuous purification effect on SRP was poor in the long term. The purification abilities of soil in the short and long terms were weak. Under intermittent operations across different seasons, SRP removal was unstable, and effluent concentration processes were different. The purification effect of the bioretention system on SRP was predicted through partial least squares regression (PLS) modeling analysis. The event mean concentration removal of SRP was positively related to the adsorption capacity of filler and rainfall interval time and negatively related to submerged zones, influent concentration and volume. PMID:29742120

Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak's extracts

NASA Astrophysics Data System (ADS)

Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat, Suzery, Meiny

2015-12-01

Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak's extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gels were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r2=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak's extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.

Purification of boron nitride nanotubes via polymer wrapping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Jin-Hyuk; Kim, Jaewoo; WCI Quantum Beam based Radiation Research Center, Korea Atomic Energy Research Institute, 1045 Daedukdaero, Daejeon 305-353

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 monitoredmore » 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.« less

Feasibility Study on Manufacturing Lightweight Aggregates from Water Purification Sludge

NASA Astrophysics Data System (ADS)

Peng, Ching-Fang; Chen, How-Ji

2018-02-01

This study mainly discussed the feasibility of manufacturing lightweight aggregates from water purification sludge in Taiwan. They were analysed for the physical and chemical composition before the sintering test for lightweight aggregates in a laboratory. Then the physical and mechanical properties of the synthesized aggregates were assessed. The result showed that the chemical composition of sludge in the water purification plants was within the appropriate range for manufacturing lightweight aggregate as proposed in the literature. The sintering test demonstrated that the particle density of aggregates from the ten types of water purification sludge were mostly less than 1.8 g/cm3. In addition, the dry unit weight, the organic impurity, the ignition loss, and other characteristics of synthesized aggregates met the requirement of CNS standards, while its water absorption and crushing strength also fulfilled the general commercial specifications. Therefore, reclamation of water purification sludge for production of lightweight aggregate is indeed feasible.

Ice-shell purification of ice-binding proteins.

PubMed

Marshall, Craig J; Basu, Koli; Davies, Peter L

2016-06-01

Ice-affinity purification is a simple and efficient method of purifying to homogeneity both natural and recombinant ice-binding proteins. The purification involves the incorporation of ice-binding proteins into slowly-growing ice and the exclusion of other proteins and solutes. In previous approaches, the ice was grown around a hollow brass finger through which coolant was circulated. We describe here an easily-constructed apparatus that employs ice affinity purification that not only shortens the time for purification from 1-2 days to 1-2 h, but also enhances yield and purity. In this apparatus, the surface area for the separation was increased by extracting the ice-binding proteins into an ice-shell formed inside a rotating round-bottom flask partially submerged in a sub-zero bath. In principle, any ice-binding compound can be recovered from liquid solution, and the method is readily scalable. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of Military Field-Water Quality. Volume 8. Performance of Mobile Water-Purification Unit (MWPU) and Pretreatment Components of the 600-GPH Reverse Osmosis Water Purification Unit (ROWPU), and Consideration of Reverse Osmosis (RO) Bypass, Potable-Water Disinfection, and Water-Quality Analysis Techniques

DTIC Science & Technology

1990-05-01

Health Risks in Potential Theaters of Operation for U.S. Military Forces. The nine volumes of this study contain a comprehensive assessment of the chemical...module. The percentage of total free chlorine ( hypochlorous acid , HOCl) plus hypochlorlte ion (OClN), measured by the Model 453 membrane sensor, varies...between the performances of the 600-Sph Reverse Osmosis Water Purification Unit (ROWPU) operated in the bypass node and ’the Mobile Water Purification

Combined cooling and purification system for nuclear reactor spent fuel pit, refueling cavity, and refueling water storage tank

DOEpatents

Corletti, Michael M.; Lau, Louis K.; Schulz, Terry L.

1993-01-01

The spent fuel pit of a pressured water reactor (PWR) nuclear power plant has sufficient coolant capacity that a safety rated cooling system is not required. A non-safety rated combined cooling and purification system with redundant branches selectively provides simultaneously cooling and purification for the spent fuel pit, the refueling cavity, and the refueling water storage tank, and transfers coolant from the refueling water storage tank to the refueling cavity without it passing through the reactor core. Skimmers on the suction piping of the combined cooling and purification system eliminate the need for separate skimmer circuits with dedicated pumps.

Polyether sulfone/hydroxyapatite mixed matrix membranes for protein purification

NASA Astrophysics Data System (ADS)

Sun, Junfen; Wu, Lishun

2014-07-01

This work proposes a novel approach for protein purification from solution using mixed matrix membranes (MMMs) comprising of hydroxyapatite (HAP) inside polyether sulfone (PES) matrix. The influence of HAP particle loading on membrane morphology is studied. The MMMs are further characterized concerning permeability and adsorption capacity. The MMMs show purification of protein via both diffusion as well as adsorption, and show the potential of using MMMs for improvements in protein purification techniques. The bovine serum albumin (BSA) was used as a model protein. The properties and structures of MMMs prepared by immersion phase separation process were characterized by pure water flux, BSA adsorption and scanning electron microscopy (SEM).

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.

New trends and affinity tag designs for recombinant protein purification.

PubMed

Wood, David W

2014-06-01

Engineered purification tags can facilitate very efficient purification of recombinant proteins, resulting in high yields and purities in a few standard steps. Over the years, many different purification tags have been developed, including short peptides, epitopes, folded protein domains, non-chromatographic tags and more recently, compound multifunctional tags with optimized capabilities. Although classic proteases are still primarily used to remove the tags from target proteins, new self-cleaving methods are gaining traction as a highly convenient alternative. In this review, we discuss some of these emerging trends, and examine their potential impacts and remaining challenges in recombinant protein research. Copyright © 2014 Elsevier Ltd. All rights reserved.

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).

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells.

PubMed

Ma, Zheng; Fung, Victor; D'Orso, Iván

2017-01-26

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

Automated high throughput microscale antibody purification workflows for accelerating antibody discovery

PubMed Central

Luan, Peng; Lee, Sophia; Paluch, Maciej; Kansopon, Joe; Viajar, Sharon; Begum, Zahira; Chiang, Nancy; Nakamura, Gerald; Hass, Philip E.; Wong, Athena W.; Lazar, Greg A.

2018-01-01

ABSTRACT To rapidly find “best-in-class” antibody therapeutics, it has become essential to develop high throughput (HTP) processes that allow rapid assessment of antibodies for functional and molecular properties. Consequently, it is critical to have access to sufficient amounts of high quality antibody, to carry out accurate and quantitative characterization. We have developed automated workflows using liquid handling systems to conduct affinity-based purification either in batch or tip column mode. Here, we demonstrate the capability to purify >2000 antibodies per day from microscale (1 mL) cultures. Our optimized, automated process for human IgG1 purification using MabSelect SuRe resin achieves ∼70% recovery over a wide range of antibody loads, up to 500 µg. This HTP process works well for hybridoma-derived antibodies that can be purified by MabSelect SuRe resin. For rat IgG2a, which is often encountered in hybridoma cultures and is challenging to purify via an HTP process, we established automated purification with GammaBind Plus resin. Using these HTP purification processes, we can efficiently recover sufficient amounts of antibodies from mammalian transient or hybridoma cultures with quality comparable to conventional column purification. PMID:29494273

Automated Purification of Recombinant Proteins: Combining High-throughput with High Yield

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lin, Chiann Tso; Moore, Priscilla A.; Auberry, Deanna L.

2006-05-01

Protein crystallography, mapping protein interactions and other approaches of current functional genomics require not only purifying large numbers of proteins but also obtaining sufficient yield and homogeneity for downstream high-throughput applications. There is a need for the development of robust automated high-throughput protein expression and purification processes to meet these requirements. We developed and compared two alternative workflows for automated purification of recombinant proteins based on expression of bacterial genes in Escherichia coli: First - a filtration separation protocol based on expression of 800 ml E. coli cultures followed by filtration purification using Ni2+-NTATM Agarose (Qiagen). Second - a smallermore » scale magnetic separation method based on expression in 25 ml cultures of E.coli followed by 96-well purification on MagneHisTM Ni2+ Agarose (Promega). Both workflows provided comparable average yields of proteins about 8 ug of purified protein per unit of OD at 600 nm of bacterial culture. We discuss advantages and limitations of the automated workflows that can provide proteins more than 90 % pure in the range of 100 ug – 45 mg per purification run as well as strategies for optimization of these protocols.« less

High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.

PubMed

Song, Dongmin; Gao, Zhendong; Zhao, Liqiang; Wang, Xiangxiang; Xu, Haijin; Bai, Yanling; Zhang, Xiuming; Linder, Markus B; Feng, Hui; Qiao, Mingqiang

2016-12-01

Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH < 7.0 and temperatures >90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins. Copyright © 2016. Published by Elsevier Inc.

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.

Isolation of viral ribonucleoprotein complexes from infected cells by tandem affinity purification.

PubMed

Mayer, Daniel; Baginsky, Sacha; Schwemmle, Martin

2005-11-01

The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.

Plasmid Vectors for Proteomic Analyses in Giardia: Purification of Virulence Factors and Analysis of the Proteasome

PubMed Central

Stadelmann, Britta; Birkestedt, Sandra; Hellman, Ulf; Svärd, Staffan G.

2012-01-01

In recent years, proteomics has come of age with the development of efficient tools for purification, identification, and characterization of gene products predicted by genome projects. The intestinal protozoan Giardia intestinalis can be transfected, but there is only a limited set of vectors available, and most of them are not user friendly. This work delineates the construction of a suite of cassette-based expression vectors for use in Giardia. Expression is provided by the strong constitutive ornithine carbamoyltransferase (OCT) promoter, and tagging is possible in both N- and C-terminal configurations. Taken together, the vectors are capable of providing protein localization and production of recombinant proteins, followed by efficient purification by a novel affinity tag combination, streptavidin binding peptide–glutathione S-transferase (SBP-GST). The option of removing the tags from purified proteins was provided by the inclusion of a PreScission protease site. The efficiency and feasibility of producing and purifying endogenous recombinant Giardia proteins with the developed vectors was demonstrated by the purification of active recombinant arginine deiminase (ADI) and OCT from stably transfected trophozoites. Moreover, we describe the tagging, purification by StrepTactin affinity chromatography, and compositional analysis by mass spectrometry of the G. intestinalis 26S proteasome by employing the Strep II-FLAG–tandem affinity purification (SF-TAP) tag. This is the first report of efficient production and purification of recombinant proteins in and from Giardia, which will allow the study of specific parasite proteins and protein complexes. PMID:22611020

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tykvart, J.; Sacha, P.; Barinka, C.

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.more » 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.« less

New data on electron-beam purification of wastewater

NASA Astrophysics Data System (ADS)

Pikaev, A. K.

2002-11-01

Recent environmental applications of radiation technology, developed in the author's laboratory, are presented in this paper. They are electron-beam and coagulation purification of molasses distillery slops from distillery-produced ethyl alcohol by fermentation of plant materials, electron-beam purification of wastewater from carboxylic acids (for example, formic acid) and removal of petroleum products (diesel fuel, motor oil and residual fuel oil) from water by γ-irradiation.

2014 Salish Kootenai College Equipment Grant

DTIC Science & Technology

2016-09-14

using an appropriate purification machine. With this appropriate equipment, protein has been purified from a yeast expression system to explore the...purification machine. With this appropriate equipment, protein has been purified from a yeast expression system to explore the effects of that protein on...Scientific Progress We have successfully purified human YKL39 chilectin protein from a yeast expression system. We are currently working on purification

Filtration in the Use of Individual Water Purification Devices

DTIC Science & Technology

2006-03-01

natural water pH will increase virus retention (references 14-17). One study investigating coliphage reduction by a 0.2 µm microporous filter...Filtration in the Use of Individual Water Purification Devices Technical Information Paper #31-004-0306 PURPOSE This information paper...natural waters . This paper is intended to assist the reader in evaluating the capabilities of Individual Water Purification Devices (IWPDs) using

9. Water Purification System and Instrument Air Receiver Tank, view ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

9. Water Purification System and Instrument Air Receiver Tank, view to the south. The water purification system is visible in the right foreground of the photograph and the instrument air receiver tank is visible in the right background of the photograph. - Washington Water Power Clark Fork River Cabinet Gorge Hydroelectric Development, Powerhouse, North Bank of Clark Fork River at Cabinet Gorge, Cabinet, Bonner County, ID

Label-Free Biomarker Detection from Whole Blood

DTIC Science & Technology

2010-02-01

we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip...nanosensors to purify biomarkers of interest. This microfluidic purification chip (MPC) captures cancer biomarkers from physiological solutions and, after...assay validation experiments (Fig. 2c). As shown in Fig. 1d, after a second valve switching step transfers MPC contents to the nanosen- sor chip, the

New Ideas for an Old Enzyme: A Short, Question-Based Laboratory Project for the Purification and Identification of an Unknown LDH Isozyme

ERIC Educational Resources Information Center

Coleman, Aaron B.

2010-01-01

Enzyme purification projects are an excellent way to introduce many aspects of protein biochemistry, but can be difficult to carry out under the constraints of a typical undergraduate laboratory course. We have designed a short laboratory project for the purification and identification of an "unknown" lactate dehydrogenase (LDH) isozyme that can…

Microplate-Based Method for High-Throughput Screening (HTS) of Chromatographic Conditions Studies for Recombinant Protein Purification.

PubMed

Carvalho, Rimenys J; Cruz, Thayana A

2018-01-01

High-throughput screening (HTS) systems have emerged as important tools to provide fast and low cost evaluation of several conditions at once since it requires small quantities of material and sample volumes. These characteristics are extremely valuable for experiments with large number of variables enabling the application of design of experiments (DoE) strategies or simple experimental planning approaches. Once, the capacity of HTS systems to mimic chromatographic purification steps was established, several studies were performed successfully including scale down purification. Here, we propose a method for studying different purification conditions that can be used for any recombinant protein, including complex and glycosylated proteins, using low binding filter microplates.

[ASSESSMENT OF POTENTIAL RISK FOR CONTAMINATION OF SURFACE WATER RESERVOIRS BY PATHOGENS OF HUMAN PARASITIC DISEASES].

PubMed

Khromenkova, E P; Dimidova, L L; Dumbadze, O S; Aidinov, G T; Shendo, G L; Agirov, A Kh; Batchaev, Kh Kh

2015-01-01

Sanitary and parasitological studies of the waste effluents and surface reservoir waters were conducted in the south of Russia. The efficiency of purification of waste effluents from the pathogens of parasitic diseases was investigated in the region's sewage-purification facilities. The water of the surface water reservoirs was found to contain helminthic eggs and larvae and intestinal protozoan cysts because of the poor purification and disinfection of service fecal sewage waters. The poor purification and disinvasion of waste effluents in the region determine the potential risk of contamination of the surface water reservoirs and infection of the population with the pathogens of human parasitic diseases.

Combined cooling and purification system for nuclear reactor spent fuel pit, refueling cavity, and refueling water storage tank

DOEpatents

Corletti, M.M.; Lau, L.K.; Schulz, T.L.

1993-12-14

The spent fuel pit of a pressured water reactor (PWR) nuclear power plant has sufficient coolant capacity that a safety rated cooling system is not required. A non-safety rated combined cooling and purification system with redundant branches selectively provides simultaneously cooling and purification for the spent fuel pit, the refueling cavity, and the refueling water storage tank, and transfers coolant from the refueling water storage tank to the refueling cavity without it passing through the reactor core. Skimmers on the suction piping of the combined cooling and purification system eliminate the need for separate skimmer circuits with dedicated pumps. 1 figures.

Purification non-aqueous solution of quantum dots CdSe- CdS-ZnS from excess organic substance-stabilizer by use PE- HD membrane

NASA Astrophysics Data System (ADS)

Kosolapova, K.; Al-Alwani, A.; Gorbachev, I.; Glukhovskoy, E.

2015-11-01

Recently, a new simple method for the purification of CdSe-CdS-ZnS quantum dots by using membrane filtration, the filtration process, successfully separated the oleic acid from quantum dots through membranes purification after synthesis; purification of quantum dots is a very significant part of post synthetical treatment that determines the properties of the material. We explore the possibilities of the Langmuir-Blodgett technique to make such layers, using quantum dots as a model system. The Langmuir monolayer of quantum dots were then investigated the surface pressure-area isotherm. From isotherm, we found the surface pressure monolayer changed with time.

[Pilot-scale purification of lipopeptide from marine-derived Bacillus marinus].

PubMed

Gu, Kangbo; Guan, Cheng; Xu, Jiahui; Li, Shulan; Luo, Yuanchan; Shen, Guomin; Zhang, Daojing; Li, Yuanguang

2016-11-25

This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.

Addition of chlorine during water purification reduces iodine content of drinking water and contributes to iodine deficiency.

PubMed

Samson, L; Czegeny, I; Mezosi, E; Erdei, A; Bodor, M; Cseke, B; Burman, K D; Nagy, E V

2012-01-01

Drinking water is the major natural source of iodine in many European countries. In the present study, we examined possible sites of iodine loss during the usual water purification process.Water samples from 6 sites during the technological process were taken and analyzed for iodine content. Under laboratory circumstances, prepared iodine in water solution has been used as a model to test the effect of the presence of chlorine. Samples from the purification sites revealed that in the presence of chlorine there is a progressive loss of iodine from the water. In the chlorine concentrations employed in the purification process, 24-h chlorine exposure eliminated more than 50% of iodine when the initial iodine concentration was 250 μg/l or less. Iodine was completely eliminated if the starting concentration was 16 μg/l.We conclude that chlorine used during water purification may be a major contributor to iodine deficiency in European communities.

[Purification Technology Optimization for Saponins from Ziziphi Spinosae Semen with Macroporous Adsorption Resin by Box-Behnken Design-Response Surface Methodology].

PubMed

Zhao, Hui-ru; Ren, Zao; Liu, Chun-ye

2015-04-01

To compare the purification effect of saponins from Ziziphi Spinosae Semen with different types of macroporous adsorption resin, and to optimize its purification technology. The type of macroporous resins was optimized by static adsorption method. The optimum technological conditions of saponins from Ziziphi Spinosae Semen was screened by single factor test and Box-Behnken Design-Response Surface Methodology. AB-8 macroporous resin had better purification effect of total saponins than other resins, optimum technological parameters were as follows: column height-diameter ratio was 5: 1, the concentration of sample solution was 2. 52 mg/mL, resin adsorption quantity was 8. 915 mg/g, eluted by 3 BV water, flow rate of adsorption and elution was 2 BV/h, elution solvent was 75% ethanol, elution solvent volume was 5 BV. AB-8 macroporous resin has a good purification effect on jujuboside A. The optimized technology is stable and feasible.

Purification of functionalized DNA origami nanostructures.

PubMed

Shaw, Alan; Benson, Erik; Högberg, Björn

2015-05-26

The high programmability of DNA origami has provided tools for precise manipulation of matter at the nanoscale. This manipulation of matter opens up the possibility to arrange functional elements for a diverse range of applications that utilize the nanometer precision provided by these structures. However, the realization of functionalized DNA origami still suffers from imperfect production methods, in particular in the purification step, where excess material is separated from the desired functionalized DNA origami. In this article we demonstrate and optimize two purification methods that have not previously been applied to DNA origami. In addition, we provide a systematic study comparing the purification efficacy of these and five other commonly used purification methods. Three types of functionalized DNA origami were used as model systems in this study. DNA origami was patterned with either small molecules, antibodies, or larger proteins. With the results of our work we aim to provide a guideline in quality fabrication of various types of functionalized DNA origami and to provide a route for scalable production of these promising tools.

Phytofilter - environmental friendly solution for purification of surface plate from urbanized territories

NASA Astrophysics Data System (ADS)

Ruchkinova, O.; Shchuckin, I.

2017-06-01

Its proved, that phytofilters are environmental friendly solution of problem of purification of surface plate from urbanized territories. Phytofilters answer the nowadays purposes to systems of purification of land drainage. The main problem of it is restrictions, connecter with its use in the conditions of cold temperature. Manufactured a technology and mechanism, which provide a whole-year purification of surface plate and its storage. Experimentally stated optimal makeup of filtering load: peat, zeolite and sand in per cent of volume, which provides defined hydraulic characteristics. Stated sorbate and ion-selective volume of complex filtering load of ordered composition in dynamic conditions. Estimated dependences of exit concentrations of oil products and heavy metals on temperature by filtering through complex filtering load of ordered composition. Defined effectiveness of purification at phytofiltering installation. Fixed an influence of embryophytes on process of phytogeneration and capacity of filtering load. Recommended swamp iris, mace reed and reed grass. Manufactured phytofilter calculation methodology. Calculated economic effect from use of phytofiltration technology in comparison with traditional block-modular installations.

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). © 2015 Elsevier Inc. All rights reserved.

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Methods in elastic tissue biology: elastin isolation and purification.

PubMed

Mecham, Robert P

2008-05-01

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60-70 kDa monomer called tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin 'contaminates' are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein's extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number.

Purification and Concentration of Nanoparticles Using Diafiltration: Scientific Operating Procedure Series: SOP-P-1

DTIC Science & Technology

2015-07-01

Coupled Plasma Mass Spectroscopy (ICP-MS) analysis If the nanoparticle of choice is a metal such as gold or silver , an aliquot can be measured using USEPA...ER D C/ EL S R- 15 -4 Environmental Consequences of Nanotechnologies Purification and Concentration of Nanoparticles Using...Environmental Consequences of Nanotechnologies ERDC/EL SR-15-4 July 2015 Purification and Concentration of Nanoparticles Using Diafiltration

Semi-automated protocol for purification of Mycobacterium leprae from tissues using the gentleMACS™ Octo Dissociator.

PubMed

Williams, Diana L; Adams, Linda B; Lahiri, Ramanuj

2014-10-01

Mycobacterium leprae, etiologic agent of leprosy, is propagated in athymic nude mouse footpads (FPs). The current purification protocol is tedious and physically demanding. A simpler, semi-automated protocol was developed using gentleMACS™ Octo Dissociator. The gentleMACS protocol provided a very effective means for purification of highly viable M. leprae from tissue. Copyright © 2014. Published by Elsevier B.V.

A simple chromatographic method for purification of egg lecithin.

PubMed

Nielsen, J R

1980-06-01

Egg lecithin was purified from the CdCl2-lecithin complex by column chromatography on Alumina. The yield from 5 eggs was 2.8 g. The purified lecithin had correct chemical values for pure lecithin and a fatty acid composition similar to lecithin prepared by other methods. The method probably can be adapted for purification of other lipids containing the phosphocholine moiety and for purification of synthetic lecithin.

Purification of recombinant Aβ(1-42) and pGlu-Aβ(3-42) using preparative SDS-PAGE.

PubMed

Spahn, Claudia; Wermann, Michael; Eichentopf, Rico; Hause, Gerd; Schlenzig, Dagmar; Schilling, Stephan

2017-08-01

Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aβ(1-42) and pGlu-Aβ(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aβ fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aβ, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was  >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aβ peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Affinity-based precipitation via a bivalent peptidic hapten for the purification of monoclonal antibodies.

PubMed

Handlogten, Michael W; Stefanick, Jared F; Deak, Peter E; Bilgicer, Basar

2014-09-07

In a previous study, we demonstrated a non-chromatographic affinity-based precipitation method, using trivalent haptens, for the purification of mAbs. In this study, we significantly improved this process by using a simplified bivalent peptidic hapten (BPH) design, which enables facile and rapid purification of mAbs while overcoming the limitations of the previous trivalent design. The improved affinity-based precipitation method (ABP(BPH)) combines the simplicity of salt-induced precipitation with the selectivity of affinity chromatography for the purification of mAbs. The ABP(BPH) method involves 3 steps: (i) precipitation and separation of protein contaminants larger than immunoglobulins with ammonium sulfate; (ii) selective precipitation of the target-antibody via BPH by inducing antibody-complex formation; (iii) solubilization of the antibody pellet and removal of BPH with membrane filtration resulting in the pure antibody. The ABP(BPH) method was evaluated by purifying the pharmaceutical antibody trastuzumab from common contaminants including CHO cell conditioned media, DNA, ascites fluid, other antibodies, and denatured antibody with >85% yield and >97% purity. Importantly, the purified antibody demonstrated native binding activity to cell lines expressing the target protein, HER2. Combined, the ABP(BPH) method is a rapid and scalable process for the purification of antibodies with the potential to improve product quality while decreasing purification costs.

Peg Precipitation Coupled with Chromatography is a New and Sufficient Method for the Purification of Botulinum Neurotoxin Type B

PubMed Central

Zhao, Yao; Kang, Lin; Gao, Shan; Gao, Xing; Xin, Wenwen; Wang, Jinglin

2012-01-01

Clostridium botulinum neurotoxins are used to treat a variety of neuro-muscular disorders, as well as in cosmetology. The increased demand requires efficient methods for the production and purification of these toxins. In this study, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin. The neurotoxin was purified by polyethylene glycol (PEG) precipitation and chromatography. Based on design of full factorial experiment, 20% (w/v) PEG-6000, 4°C, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate of 87% for the type B neurotoxin complex, as indicated by a purification factor of 61.5 fold. Furthermore, residual bacterial cells, impurity proteins and some nucleic acids were removed by PEG precipitation. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl™ S-100 and phenyl HP columns. The neurotoxin was recovered with an overall yield of 21.5% and the purification factor increased to 216.7 fold. In addition, a mouse bioassay determined the purified neurotoxin complex possessed a specific toxicity (LD50) of 4.095 ng/kg. PMID:22761863

Plasmid pVAX1-NH36 purification by membrane and bead perfusion chromatography.

PubMed

Franco-Medrano, Diana Ivonne; Guerrero-Germán, Patricia; Montesinos-Cisneros, Rosa María; Ortega-López, Jaime; Tejeda-Mansir, Armando

2017-03-01

The demand for plasmid DNA (pDNA) has increased in response to the rapid advances in vaccines applications to prevent and treat infectious diseases caused by virus, bacteria or parasites, such as Leishmania species. The immunization protocols require large amounts of supercoiled plasmid DNA (sc-pDNA) challenging the development of efficient and profitable processes for capturing and purified pDNA molecules from large volumes of lysates. A typical bioprocess involves four steps: fermentation, primary recovery, intermediate recovery and final purification. Ion-exchange chromatography is one of the key operations in the purification schemes of pDNA owing the chemical structure of these macromolecules. The goal of this research was to compare the performance of the final purification step of pDNA using ion-exchange chromatography on columns packed with Mustang Q membranes or perfusive beads POROS 50 HQ. The experimental results showed that both matrixes could separate the plasmid pVAX1-NH36 (3936 bp) from impurities in clarified Escherichia coli lysates with an adequate resolution. In addition, a 24- and 21-fold global purification factor was obtained. An 88 and 63% plasmid recuperation was achieved with ion-exchange membranes and perfusion beads, respectively. A better understanding of perfusion-based matrices for the purification of pDNA was developed in this research.

Single-step affinity purification of enzyme biotherapeutics: a platform methodology for accelerated process development.

PubMed

Brower, Kevin P; Ryakala, Venkat K; Bird, Ryan; Godawat, Rahul; Riske, Frank J; Konstantinov, Konstantin; Warikoo, Veena; Gamble, Jean

2014-01-01

Downstream sample purification for quality attribute analysis is a significant bottleneck in process development for non-antibody biologics. Multi-step chromatography process train purifications are typically required prior to many critical analytical tests. This prerequisite leads to limited throughput, long lead times to obtain purified product, and significant resource requirements. In this work, immunoaffinity purification technology has been leveraged to achieve single-step affinity purification of two different enzyme biotherapeutics (Fabrazyme® [agalsidase beta] and Enzyme 2) with polyclonal and monoclonal antibodies, respectively, as ligands. Target molecules were rapidly isolated from cell culture harvest in sufficient purity to enable analysis of critical quality attributes (CQAs). Most importantly, this is the first study that demonstrates the application of predictive analytics techniques to predict critical quality attributes of a commercial biologic. The data obtained using the affinity columns were used to generate appropriate models to predict quality attributes that would be obtained after traditional multi-step purification trains. These models empower process development decision-making with drug substance-equivalent product quality information without generation of actual drug substance. Optimization was performed to ensure maximum target recovery and minimal target protein degradation. The methodologies developed for Fabrazyme were successfully reapplied for Enzyme 2, indicating platform opportunities. The impact of the technology is significant, including reductions in time and personnel requirements, rapid product purification, and substantially increased throughput. Applications are discussed, including upstream and downstream process development support to achieve the principles of Quality by Design (QbD) as well as integration with bioprocesses as a process analytical technology (PAT). © 2014 American Institute of Chemical Engineers.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yacoby, I.; Tegler, L. T.; Pochekailov, S.

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,more » 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.« less

Regenerated silica gel as stationary phase on vacuum column chromatography to purify temulawak’s extracts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cahyono, Bambang; Maduwu, Ratna Dewi; Widayat,

Commercial silica gel only used once by many researchers and affected high cost for purification process, also less support the green chemistry program. This research focused in regeneration silica gel that used purification of temulawak’s extracts (Curcuma xanthorrhiza Roxb) by vacuum column chromatography. Sample extracts (contains 10.1195±0.5971% of curcuminoids) was purified by vacuum column chromatography (pressure: 45 kPa, column: 100mm on length and 16mm on diameter). Ethanol 96% and acetone were compared as eluent. The amount of solvent and yield of curcuminoids used as indicator purification. The silica gel was regenerated with heating in 600°C for 8 hours The silica gelsmore » were analyzed by IR spectroscopy and X-ray diffraction. Furthermore, regenerated silica gel was used as the stationary phase in vacuum column chromatography under the same conditions with the previous purification. All the purification experiments were performed in three repetitions. Based on regression equation, y=0.132x+0.0011 (r{sup 2}=0.9997) the yield of curcuminoids on purified products using ethanol as the eluent was improved 4.26% (to 14.3724±0.5749%) and by acetone was improved 3,03% (to 13.1450 ±0.6318%). The IR spectrum of both silica gel showed the same vibration profile and also there were three crystallinity peaks missing on its X-ray diffraction. Regenerated silica gel has the same performance with new silica gel in purification of temulawak’s extract: by ethanol has increased 4.08% (14.1947±0.7415%) and 2.93% (13.0447±0.4822) by acetone. In addition, all purification products showed similar TLC profiles. Purification using regenerated silica gel as the adsorbent on vacuum column chromatography has exactly same potential with the new silica gel.« less

Analysis And Design Of A Water Purification System For The West African Area Of Operation

DTIC Science & Technology

2016-12-01

harmful metals and in disinfecting the water prior to human consumption . Research conducted proved that the BWS is more cost effective , efficient...and test a feasible and cost- effective prototype of a purification system to the BWS for improved capability. This study uses a design-based and...design. The prototype test results showed that the water purification system performed effectively and efficiently in accordance with the

An automated synthesis-purification-sample-management platform for the accelerated generation of pharmaceutical candidates.

PubMed

Sutherland, J David; Tu, Noah P; Nemcek, Thomas A; Searle, Philip A; Hochlowski, Jill E; Djuric, Stevan W; Pan, Jeffrey Y

2014-04-01

A flexible and integrated flow-chemistry-synthesis-purification compound-generation and sample-management platform has been developed to accelerate the production of small-molecule organic-compound drug candidates in pharmaceutical research. Central to the integrated system is a Mitsubishi robot, which hands off samples throughout the process to the next station, including synthesis and purification, sample dispensing for purity and quantification analysis, dry-down, and aliquot generation.

Tentacle-type immobilized metal affinity cryogel for invertase purification from Saccharomyces cerevisiae.

PubMed

Çetin, Kemal; Perçin, Işık; Denizli, Fatma; Denizli, Adil

2017-11-01

The aim of this study is to investigate the usability of cryogel columns for the purification of invertase from Saccharomyces cerevisiae. Poly(2-hydroxyethyl methacrylate) monolithic columns were produced via cryogelation. Ester groups of the poly(2-hydroxyethyl methacrylate) structure were then converted to imine groups by the reaction with poly(ethylene imine) in the presence of NaHCO 3 . Transition metal ions, Cu(II), Co(II), and Ni(II), were chelated on the PEI-modified cryogel columns. Purification of invertase from natural source namely S. cerevisiae was also studied, and the purification fold values were obtained as 41.350, 44.714, and 30.302 for Cu(II)-chelated, Co(II)-chelated, and Ni(II)-chelated PHEMA/PEI columns, respectively.

Recent Advances in Nanoporous Membranes for Water Purification

PubMed Central

Wang, Zhuqing; Colombi Ciacchi, Lucio

2018-01-01

Nanoporous materials exhibit wide applications in the fields of electrocatalysis, nanodevice fabrication, energy, and environmental science, as well as analytical science. In this review, we present a summary of recent studies on nanoporous membranes for water purification application. The types and fabrication strategies of various nanoporous membranes are first introduced, and then the fabricated nanoporous membranes for removing various water pollutants, such as salt, metallic ions, anions, nanoparticles, organic chemicals, and biological substrates, are demonstrated and discussed. This work will be valuable for readers to understand the design and fabrication of various nanoporous membranes, and their potential purification mechanisms towards different water pollutants. In addition, it will be helpful for developing new nanoporous materials for quick, economic, and high-performance water purification. PMID:29370128

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography.

PubMed

Gao, Min; Gu, Ming; Liu, Chun-Zhao

2006-07-11

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.

Bromelain: an overview of industrial application and purification strategies.

PubMed

Arshad, Zatul Iffah Mohd; Amid, Azura; Yusof, Faridah; Jaswir, Irwandi; Ahmad, Kausar; Loke, Show Pau

2014-09-01

This review highlights the use of bromelain in various applications with up-to-date literature on the purification of bromelain from pineapple fruit and waste such as peel, core, crown, and leaves. Bromelain, a cysteine protease, has been exploited commercially in many applications in the food, beverage, tenderization, cosmetic, pharmaceutical, and textile industries. Researchers worldwide have been directing their interest to purification strategies by applying conventional and modern approaches, such as manipulating the pH, affinity, hydrophobicity, and temperature conditions in accord with the unique properties of bromelain. The amount of downstream processing will depend on its intended application in industries. The breakthrough of recombinant DNA technology has facilitated the large-scale production and purification of recombinant bromelain for novel applications in the future.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Ultrasound-assisted three-phase partitioning of polyphenol oxidase from potato peel (Solanum tuberosum).

PubMed

Niphadkar, Sonali S; Rathod, Virendra K

2015-01-01

Conventional three phase partitioning (TPP) and ultrasound assisted three phase partitioning (UATPP) were optimized for achieving the maximum extraction and purification of polyphenol oxidase (PPO) from waste potato peels. Different process parameters such as ammonium sulfate (NH4)2SO4 concentration, crude extract to t-butanol ratio, time, temperature and pH were studied for conventional TPP. Except agitation speed, the similar parameters were also optimized for UATPP. Further additional parameters were also studied for UATPP viz. irradiation time at different frequencies, duty cycle and, rated power in order to obtain the maximum purification factor and recovery of PPO. The optimized conditions for conventional TPP were (NH4)2SO4 0-40% (w/v), extract to t-butanol ratio 1:1 (v/v), time 40 min and pH 7 at 30°C. These conditions provided 6.3 purification factor and 70% recovery of PPO from bottom phase. On the other hand, UATPP gives maximum purification fold of 19.7 with 98.3% recovery under optimized parameters which includes (NH4)2SO4 0-40% (w/v), crude extract to t-butanol ratio 1: 1 (v/v) pH 7, irradiation time 5 min with 25 kHz, duty cycle 40% and rated power 150W at 30°C. UATPP delivers higher purification factor and % recovery of PPO along with reduced operation time from 40 min to 5 min when compared with TPP. SDS PAGE showed partial purification of PPO enzyme with UATPP with molecular weight in the range of 26-36 kDa. Results reveal that UATPP would be an attractive option for the isolation and purification of PPO without need of multiple steps. © 2015 American Institute of Chemical Engineers.

Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag.

PubMed

Li, Junhua; Zhang, Yang; Yang, Yanjun

2013-03-01

The ribosomal protein L2, a constituent protein of the 50S large ribosomal subunit, can be used as Si-tag using silica particles for the immobilization and purification of recombinant proteins (Ikeda et al. (Protein Expr Purif 71:91-95, 2010); Taniguchi et al. (Biotechnol Bioeng 96:1023-1029, 2007)). We applied a diatomite powder, a sedimentary rock mainly composed with diatoms silica, as an affinity solid phase and small ubiquitin-like modifier (SUMO) technology to release a target protein from the solid phase. The L2 (203-273) was the sufficient region for the adsorption of ribosomal protein L2 on diatomite. We comparatively analyzed the different adsorption properties of the two deleted proteins of L2 (L2 (1-60, 203-273) and L2 (203-273)) on diatomite. The time required to reach adsorption equilibrium of L2 (203-273) fusion protein on diatomite was shorter than that of L2 (1-60, 203-273) fusion protein. The maximum adsorption capacity of L2 (203-273) fusion protein was larger than that of L2 (1-60, 203-273) fusion protein. In order to study whether the L2 (203-273) can function as an affinity purification tag, SUMO was introduced as one specific protease cleavage site between the target protein and the purification tags. The L2 (203-273) and SUMO fusion protein purification method was tested using enhanced green fluorescent protein as a model protein; the result shows that the purification performance of this affinity purification method was good. The strong adsorption characteristic of L2 (203-273) on diatomite also provides a potential protein fusion tag for the immobilization of enzyme.

Semi-automated hydrophobic interaction chromatography column scouting used in the two-step purification of recombinant green fluorescent protein.

PubMed

Stone, Orrin J; Biette, Kelly M; Murphy, Patrick J M

2014-01-01

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. 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. 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 development other of HIC-compatible protein purification schemes.

Protein body-inducing fusions for high-level production and purification of recombinant proteins in plants.

PubMed

Conley, Andrew J; Joensuu, Jussi J; Richman, Alex; Menassa, Rima

2011-05-01

For the past two decades, therapeutic and industrially important proteins have been expressed in plants with varying levels of success. The two major challenges hindering the economical production of plant-made recombinant proteins include inadequate accumulation levels and the lack of efficient purification methods. To address these limitations, several fusion protein strategies have been recently developed to significantly enhance the production yield of plant-made recombinant proteins, while simultaneously assisting in their subsequent purification. Elastin-like polypeptides are thermally responsive biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the purification of recombinant proteins. Hydrophobins are small fungal proteins capable of altering the hydrophobicity of their respective fusion partner, thus enabling efficient purification by surfactant-based aqueous two-phase systems. Zera, a domain of the maize seed storage protein γ-zein, can induce the formation of protein storage bodies, thus facilitating the recovery of fused proteins using density-based separation methods. These three novel protein fusion systems have also been shown to enhance the accumulation of a range of different recombinant proteins, while concurrently inducing the formation of protein bodies. The packing of these fusion proteins into protein bodies may exclude the recombinant protein from normal physiological turnover. Furthermore, these systems allow for quick, simple and inexpensive nonchromatographic purification of the recombinant protein, which can be scaled up to industrial levels of protein production. This review will focus on the similarities and differences of these artificial storage organelles, their biogenesis and their implication for the production of recombinant proteins in plants and their subsequent purification. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Optimization of Ammonium Sulfate Concentration for Purification of Colorectal Cancer Vaccine Candidate Recombinant Protein GA733-FcK Isolated from Plants.

PubMed

Park, Se-Ra; Lim, Chae-Yeon; Kim, Deuk-Su; Ko, Kisung

2015-01-01

A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25-30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. Thus, the objective in this study is to optimize the conditions for plant-derived protein purification procedures. Various ammonium sulfate concentrations (15-80%) were compared to determine their effects on TSPs yield. With 50% ammonium sulfate, the yield of precipitated TSP was the highest, and that of the plant-derived colorectal cancer-specific surface glycoprotein GA733 fused to the Fc fragment of human IgG tagged with endoplasmic reticulum retention signal KDEL (GA733(P)-FcK) protein significantly increased 1.8-fold. SDS-PAGE analysis showed that the purity of GA733(P)-FcK protein band appeared to be similar to that of an equal dose of mammalian-derived GA733-Fc (GA733(M)-Fc). The binding activity of purified GA733(P)-FcK to anti-GA733 mAb was as efficient as the native GA733(M)-Fc. Thus, the purification process was effectively optimized for obtaining a high yield of plant-derived antigenic protein with good quality. In conclusion, the purification recovery rate of large quantities of recombinant protein from plant expression systems can be enhanced via optimization of ammonium sulfate concentration during downstream processes, thereby offering a promising solution for production of recombinant GA733-Fc protein in plants.

Preparation and Purification of Multigram Quantities of TAX and SEX.

DTIC Science & Technology

1981-12-01

Synthesis Purification Nitrolysis 2L AS[TNIACT (Cletiloe -m powowe0m N noeaemy and Identify by block number) This final report describes the multigram... synthesis and purification of 3 kg of 1-acetylhexahydro-3,5-dinitro-1,3,5-triazine (TAX) and the feasibility of producing kilogram quantities of l...residual impurities; (3) demonstrate the feasibility of the synthesis approach on a one-pound batch reaction; and (4) provide a cost-plus-fixed-fee estimate

Methods for Isolation, Purification, and Propagation of Bacteriophages of Campylobacter jejuni.

PubMed

Gencay, Yilmaz Emre; Birk, Tina; Sørensen, Martine Camilla Holst; Brøndsted, Lone

2017-01-01

Here, we describe the methods for isolation, purification, and propagation of Campylobacter jejuni bacteriophages from samples expected to contain high number of phages such as chicken feces. The overall steps are (1) liberation of phages from the sample material; (2) observation of plaque-forming units on C. jejuni lawns using a spot assay; (3) isolation of single plaques; (4) consecutive purification procedures; and (5) propagation of purified phages from a plate lysate to prepare master stocks.

Sensitivity of measurement-based purification processes to inner interactions

NASA Astrophysics Data System (ADS)

Militello, Benedetto; Napoli, Anna

2018-02-01

The sensitivity of a repeated measurement-based purification scheme to additional undesired couplings is analyzed, focusing on the very simple and archetypical system consisting of two two-level systems interacting with a repeatedly measured one. Several regimes are considered and in the strong coupling limit (i.e., when the coupling constant of the undesired interaction is very large) the occurrence of a quantum Zeno effect is proven to dramatically jeopardize the efficiency of the purification process.

Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

DTIC Science & Technology

2005-01-01

13-09-20061 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cloning, expression and purification of Brucella suis outer membrane proteins 5b. GRANT NUMBER...attractive for this purpose. In this study, we cloned, expressed and purified seven predicted OMPs of Brucella suis . The recombinant proteins were...fused with 6-his and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based

The purification process on scintillator material (SrI{sub 2}: Eu) by zone-refinement technique

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arumugam, Raja; Daniel, D. Joseph; Ramasamy, P., E-mail: ramasamyp@ssn.edu.in

The thermal properties of Europium doped strontium iodide was analyzed through Thermogravimetric (TG) and differential thermal analyses (DTA). The melting point of europium doped strontium iodide is around 531°C. The hydrated and oxyhalide impurities were found before melting temperature. In order to remove these impurities we have done purification process by Zone-refinement technique. The effective output of purification of zone refining was also observed through the segregation of impurities.

Purification of swine haptoglobin by affinity chromatography.

PubMed Central

Eurell, T E; Hall, W F; Bane, D P

1990-01-01

A globin-agarose affinity chromatography technique was used to purify swine haptoglobin. This technique provides a highly specific, single-step purification method without the contamination of extraneous serum proteins reported by previous studies. Complex formation between the haptoglobin isolate and swine hemoglobin confirmed that biological activity was maintained during the purification process. Immunoelectrophoretic and Ouchterlony immunodiffusion methods revealed that the swine haptoglobin isolate cross-reacted with polyvalent antisera against human haptoglobin. Images Fig. 2. Fig. 3. PMID:2123414

Comparison of two methods for purification of enterocin B, a bacteriocin produced by Enterococcus faecium W3.

PubMed

Dündar, Halil; Atakay, Mehmet; Çelikbıçak, Ömür; Salih, Bekir; Bozoğlu, Faruk

2015-01-01

This study aimed to compare two different approaches for the purification of enterocin B from Enterococcus faecium strain W3 based on the observation that the bacteriocin was found both in cell associated form and in culture supernatant. The first approach employed ammonium sulfate precipitation, cation-exchange chromatography, and sequential reverse-phase high-performance liquid chromatography. The latter approach exploited a pH-mediated cell adsorption-desorption method to extract cell-bound bacteriocin, and one run of reverse-phase chromatography. The first method resulted in purification of enterocin B with a recovery of 4% of the initial bacteriocin activity found in culture supernatant. MALDI-TOF MS analysis and de novo peptide sequencing of the purified bacteriocin confirmed that the active peptide was enterocin B. The second method achieved the purification of enterocin B with a higher recovery (16%) and enabled us to achieve pure bacteriocin within a shorter period of time by avoiding time consuming purification protocols. The purity and identity of the active peptide were confirmed again by matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry (MS) analysis. Although both approaches were satisfactory to obtain a sufficient amount of enterocin B for use in MS and amino acid sequence analysis, the latter was proved to be applicable in large-scale and rapid purification of enterocin B.

Advanced purification of petroleum refinery wastewater by catalytic vacuum distillation.

PubMed

Yan, Long; Ma, Hongzhu; Wang, Bo; Mao, Wei; Chen, Yashao

2010-06-15

In our work, a new process, catalytic vacuum distillation (CVD) was utilized for purification of petroleum refinery wastewater that was characteristic of high chemical oxygen demand (COD) and salinity. Moreover, various common promoters, like FeCl(3), kaolin, H(2)SO(4) and NaOH were investigated to improve the purification efficiency of CVD. Here, the purification efficiency was estimated by COD testing, electrolytic conductivity, UV-vis spectrum, gas chromatography-mass spectrometry (GC-MS) and pH value. The results showed that NaOH promoted CVD displayed higher efficiency in purification of refinery wastewater than other systems, where the pellucid effluents with low salinity and high COD removal efficiency (99%) were obtained after treatment, and the corresponding pH values of effluents varied from 7 to 9. Furthermore, environment estimation was also tested and the results showed that the effluent had no influence on plant growth. Thus, based on satisfied removal efficiency of COD and salinity achieved simultaneously, NaOH promoted CVD process is an effective approach to purify petroleum refinery wastewater. Copyright 2010 Elsevier B.V. All rights reserved.

General method for rapid purification of native chromatin fragments.

PubMed

Kuznetsov, Vyacheslav I; Haws, Spencer A; Fox, Catherine A; Denu, John M

2018-05-24

Biochemical, proteomic and epigenetic studies of chromatin rely on the efficient ability to isolate native nucleosomes in high yield and purity. However, isolation of native chromatin suitable for many downstream experiments remains a challenging task. This is especially true for the budding yeast Saccharomyces cerevisiae, which continues to serve as an important model organism for the study of chromatin structure and function. Here, we developed a time- and cost-efficient universal protocol for isolation of native chromatin fragments from yeast, insect, and mammalian cells. The resulting protocol preserves histone posttranslational modification in the native chromatin state, and is applicable for both parallel multi-sample spin-column purification and large scale isolation. This protocol is based on the efficient and stable purification of polynucleosomes, features a combination of optimized cell lysis and purification conditions, three options for chromatin fragmentation, and a novel ion-exchange chromatographic purification strategy.  The procedure will aid chromatin researchers interested in isolating native chromatin material for biochemical studies, and as a mild, acid- and detergent-free sample preparation method for mass-spectrometry analysis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Purification of plant plasma membranes by two-phase partitioning and measurement of H+ pumping.

PubMed

Lund, Anette; Fuglsang, Anja Thoe

2012-01-01

Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.Purification of plasma membranes by two-phase partitioning is based on the separation of microsomal membranes, dependent on their surface hydrophobicity. Here we explain the purification of plasma membranes from a relatively small amount of material (7-30 g). The fluorescent probe ACMA (9-amino-6-chloro-2-metoxyacridine) accumulates inside the vesicles upon protonation. Quenching of ACMA in the solution corresponds to the H(+) transport across the plasma membrane. Before running the assay, the plasma membranes are incubated with the detergent Brij-58 in order to create inside-out vesicles.

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.

Methods in Elastic Tissue Biology: Elastin Isolation and Purification

PubMed Central

Mecham, Robert P.

2008-01-01

Elastin provides recoil to tissues subjected to repeated stretch, such as blood vessels and the lung. It is encoded by a single gene in mammals and is secreted as a 60–70 kDa monomer call tropoelastin. The functional form of the protein is that of a large, highly crosslinked polymer that organizes as sheets or fibers in the extracellular matrix. Purification of mature, crosslinked elastin is problematic because its insolubility precludes its isolation using standard wet-chemistry techniques. Instead, relatively harsh experimental approaches designed to remove non-elastin ‘contaminates’ are employed to generate an insoluble product that has the amino acid composition expected of elastin. Although soluble, tropoelastin also presents problems for isolation and purification. The protein’s extreme stickiness and susceptibility to proteolysis requires careful attention during purification and in tropoelastin-based assays. This article describes the most common approaches for purification of insoluble elastin and tropoelastin. It also addresses key aspects of studying tropoelastin production in cultured cells, where elastin expression is highly dependent upon cell type, culture conditions, and passage number. PMID:18442703

Purification of infectious adenovirus in two hours by ultracentrifugation and tangential flow filtration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ugai, Hideyo; Yamasaki, Takahito; Hirose, Megumi

2005-06-17

Adenoviruses are excellent vectors for gene transfer and are used extensively for high-level expression of the products of transgenes in living cells. The development of simple and rapid methods for the purification of stable infectious recombinant adenoviruses (rAds) remains a challenge. We report here a method for the purification of infectious adenovirus type 5 (Ad5) that involves ultracentrifugation on a cesium chloride gradient at 604,000g for 15 min at 4 deg C and tangential flow filtration. The entire procedure requires less than two hours and infectious Ad5 can be recovered at levels higher than 64% of the number of plaque-formingmore » units (pfu) in the initial crude preparation of viruses. We have obtained titers of infectious purified Ad5 of 1.35 x 10{sup 10} pfu/ml and a ratio of particle titer to infectious titer of seven. The method described here allows the rapid purification of rAds for studies of gene function in vivo and in vitro, as well as the rapid purification of Ad5.« less

Direct purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel using a PEG/salt-based Aqueous Two Phase System.

PubMed

Mehrnoush, Amid; Sarker, Md Zaidul Islam; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

2011-10-10

An Aqueous Two-Phase System (ATPS) was employed for the first time for the separation and purification of pectinase from mango (Mangifera Indica Cv. Chokanan) peel. The effects of different parameters such as molecular weight of the polymer (polyethylene glycol, 2,000-10,000), potassium phosphate composition (12-20%, w/w), system pH (6-9), and addition of different concentrations of neutral salts (0-8%, w/w) on partition behavior of pectinase were investigated. The partition coefficient of the enzyme was decreased by increasing the PEG molecular weight. Additionally, the phase composition showed a significant effect on purification factor and yield of the enzyme. Optimum conditions for purification of pectinase from mango peel were achieved in a 14% PEG 4000-14% potassium phosphate system using 3% (w/w) NaCl addition at pH 7.0. Based on this system, the purification factor of pectinase was increased to 13.2 with a high yield of (97.6%). Thus, this study proves that ATPS can be an inexpensive and effective method for partitioning of pectinase from mango peel.

Purification of pectinase from mango (Mangifera indica L. cv. Chokanan) waste using an aqueous organic phase system: a potential low cost source of the enzyme.

PubMed

Amid, Mehrnoush; Abdul Manap, Mohd Yazid; Mustafa, Shuhaimi

2013-07-15

As a novel method of purification, an aqueous organic phase system (AOPS) was employed to purify pectinase from mango waste. The effect of different parameters, such as the alcohol concentration (ethanol, 1-propanol, and 2-propanol), the salt type and concentration (ammonium sulfate, potassium phosphate and sodium citrate), the feed stock crude load, the aqueous phase pH and NaCl concentration, were investigated in the recovery of pectinase from mango peel. The partition coefficient (K), selectivity (S), purification factor (PF) and yield (Y, %) were investigated in this study as important parameters for the evaluation of enzyme recovery. The desirable partition efficiency for pectinase purification was achieved in an AOPS of 19% (w/w) ethanol and 22% (w/w) potassium phosphate in the presence of 5% (w/w) NaCl at pH 7.0. Based on the system, the purification factor of pectinase was enhanced 11.7, with a high yield of 97.1%. Copyright © 2013 Elsevier B.V. All rights reserved.

Tab2, a novel recombinant polypeptide tag offering sensitive and specific protein detection and reliable affinity purification.

PubMed

Crusius, Kerstin; Finster, Silke; McClary, John; Xia, Wei; Larsen, Brent; Schneider, Douglas; Lu, Hong-Tao; Biancalana, Sara; Xuan, Jian-Ai; Newton, Alicia; Allen, Debbie; Bringmann, Peter; Cobb, Ronald R

2006-10-01

The detection and purification of proteins are often time-consuming and frequently involve complicated protocols. The addition of a peptide tag to recombinant proteins can make this process more efficient. Many of the commonly used tags, such as Flagtrade mark, Myc, HA and V5 are recognized by specific monoclonal antibodies and therefore, allow immunoaffinity-based purification. Enhancing the current scope of flexibility in using diverse peptide tags, we report here the development of a novel, short polypeptide tag (Tab2) for detection and purification of recombinant proteins. The Tab2 epitope corresponds to the NH2-terminal seven amino acid residues of human TGFalpha. A monoclonal anti-Tab2 antibody was raised and characterized. To investigate the potential of this peptide sequence as a novel tag for recombinant proteins, we expressed several different recombinant proteins containing this tag in E. coli, baculovirus, and mammalian cells. The data presented demonstrates the Tab2 tag-anti-Tab2 antibody combination is a reliable tool enabling specific Western blot detection, FACS analysis, and immunoprecipitation as well as non-denaturing protein affinity purification.

Very large scale monoclonal antibody purification: the case for conventional unit operations.

PubMed

Kelley, Brian

2007-01-01

Technology development initiatives targeted for monoclonal antibody purification may be motivated by manufacturing limitations and are often aimed at solving current and future process bottlenecks. A subject under debate in many biotechnology companies is whether conventional unit operations such as chromatography will eventually become limiting for the production of recombinant protein therapeutics. An evaluation of the potential limitations of process chromatography and filtration using today's commercially available resins and membranes was conducted for a conceptual process scaled to produce 10 tons of monoclonal antibody per year from a single manufacturing plant, a scale representing one of the world's largest single-plant capacities for cGMP protein production. The process employs a simple, efficient purification train using only two chromatographic and two ultrafiltration steps, modeled after a platform antibody purification train that has generated 10 kg batches in clinical production. Based on analyses of cost of goods and the production capacity of this very large scale purification process, it is unlikely that non-conventional downstream unit operations would be needed to replace conventional chromatographic and filtration separation steps, at least for recombinant antibodies.

A comparative study of different strategies for removal of endotoxins from bacteriophage preparations.

PubMed

Van Belleghem, Jonas D; Merabishvili, Maya; Vergauwen, Bjorn; Lavigne, Rob; Vaneechoutte, Mario

2017-01-01

Bacterial endotoxins have high immunogenicity. Phage biology studies as well as therapeutic phage applications necessitate highly purified phage particles. In this study, we compared combinations of seven different endotoxin removal strategies and validated their endotoxin removal efficacy for five different phages (i.e. four Pseudomonas aeruginosa phages and one Staphylococcus aureus phage). These purification strategies included Endotrap HD column purification and/or CsCl density centrifugation in combination with Endotrap purification, followed by organic solvent (1-octanol), detergent (Triton X-100), enzymatic inactivation of the endotoxin using alkaline phosphatase and CIM monolytic anion exchange chromatography. We show that CsCl density purification of the P. aeruginosa phages, at an initial concentration of 10 12 -10 13 pfu/ml, led to the strongest reduction of endotoxins, with an endotoxin removal efficacy of up to 99%, whereas additional purification methods did not result in a complete removal of endotoxins from the phage preparations and only yielded an additional endotoxin removal efficacy of 23 to 99%, sometimes accompanied with strong losses in phage titer. Copyright © 2016 Elsevier B.V. All rights reserved.

Partial purification of penicillin acylase from Escherichia coli in poly(ethylene glycol)-sodium citrate aqueous two-phase systems.

PubMed

Marcos, J C; Fonseca, L P; Ramalho, M T; Cabral, J M

1999-10-29

Studies on the partition and purification of penicillin acylase from Escherichia coli osmotic shock extract were performed in poly(ethylene glycol)-sodium citrate systems. Partition coefficient behavior of the enzyme and total protein are similar to those described in other reports, increasing with pH and tie line length and decreasing with PEG molecular weight. However, some selectivity could be attained with PEG 1000 systems and long tie line at pH 6.9. Under these conditions 2.6-fold purification with 83% yield were achieved. Influence of pH on partition shows that is the composition of the system and not the net charge of the enzyme that determines the behaviour in these conditions. Addition of NaCl to PEG 3350 systems significantly increases the partition of the enzyme. Although protein partition also increased, purification conditions were possible with 1.5 M NaCl where 5.7-fold purification and 85% yield was obtained. This was possible due to the higher hydrophobicity of the enzyme compared to that of most contaminants proteins.

Automated multi-dimensional purification of tagged proteins.

PubMed

Sigrell, Jill A; Eklund, Pär; Galin, Markus; Hedkvist, Lotta; Liljedahl, Pia; Johansson, Christine Markeland; Pless, Thomas; Torstenson, Karin

2003-01-01

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. AKTA 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His6- or GST-tagged proteins per day and can produce 1-50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.

Automated large-scale purification of a G protein-coupled receptor for neurotensin.

PubMed

White, Jim F; Trinh, Loc B; Shiloach, Joseph; Grisshammer, Reinhard

2004-04-30

Structure determination of integral membrane proteins requires milligram amounts of purified, functional protein on a regular basis. Here, we describe a protocol for the purification of a G protein-coupled neurotensin receptor fusion protein at the 3-mg or 10-mg level using immobilized metal affinity chromatography and a neurotensin column in a fully automated mode. Fermentation at a 200-l scale of Escherichia coli expressing functional receptors provides the material needed to feed into the purification routine. Constructs with tobacco etch virus protease recognition sites at either end of the receptor allow the isolation of neurotensin receptor devoid of its fusion partners. The presented expression and purification procedures are simple and robust, and provide the basis for crystallization experiments of receptors on a routine basis.

Recombinant protein expression and purification: a comprehensive review of affinity tags and microbial applications.

PubMed

Young, Carissa L; Britton, Zachary T; Robinson, Anne S

2012-05-01

Protein fusion tags are indispensible tools used to improve recombinant protein expression yields, enable protein purification, and accelerate the characterization of protein structure and function. Solubility-enhancing tags, genetically engineered epitopes, and recombinant endoproteases have resulted in a versatile array of combinatorial elements that facilitate protein detection and purification in microbial hosts. In this comprehensive review, we evaluate the most frequently used solubility-enhancing and affinity tags. Furthermore, we provide summaries of well-characterized purification strategies that have been used to increase product yields and have widespread application in many areas of biotechnology including drug discovery, therapeutics, and pharmacology. This review serves as an excellent literature reference for those working on protein fusion tags. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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.

New Contribution to the Method of Van Arkel for the Purification of Metals on Incandescent Filaments; NUEVAS APORTACIONES AL METODO DE VAN ARKEL PARA LA PURIFICACION DE METALES SOBRE FILAMENTOS INCANDESCENTES

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodriguez, M.L.; Martorell, J.T.

1962-01-01

The purification of zirconium in a cyclical static process using ZrI/sub 4/ as the volatile compound and W filaments was studied after a review of previous works on the subject. The equations corresponding to the isothermal process are given, in some detail. The optimum conditions of temperature and velocity for the maximum purification of the metal were determined. (J.S.R.)

Water purification in Borexino

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giammarchi, M.; Balata, M.; Ioannucci, L.

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.

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.

Experimental Study on Purification of Low Grade Diatomite

NASA Astrophysics Data System (ADS)

Xiao, Liguang; Pang, Bo

2017-04-01

This paper presented an innovation for purification of low grade diatomite(DE) by grinding, ultrasonic pretreatment, acid leaching of closed stirring and calcination. The optimum process parameters of DE purification were obtained, the characterizations of original and purified DE were determined by SEM and BET. The results showed that the specific surface area of DE increased from 12.65m2/g to 23.23m2/g, which increased by 45.54%. SEM analysis revealed that the pore structure of purified DE was dredged highly.

Comparative aspects of the purification and properties of cholinesterases

PubMed Central

Augustinsson, Klas-Bertil

1971-01-01

Recent years have seen great progress in the purification and characterization of cholinesterases. Investigation has indicated the existence of two principal groups: a fairly homogeneous group of acetylcholinesterases and a group of enzymes that utilize butyrylcholine, propionycholine, or benzoylcholine as substrates and that differ widely in their properties. This paper reviews the different types of cholinesterase and their sources, the importance of a proper choice of substrate in cholinesterase studies, methods for the purification of cholinesterases, and some of the properties of these enzymes. PMID:4938026

Recovery and purification of ethylene

DOEpatents

Reyneke, Rian [Katy, TX; Foral, Michael J [Aurora, IL; Lee, Guang-Chung [Houston, TX; Eng, Wayne W. Y. [League City, TX; Sinclair, Iain [Warrington, GB; Lodgson, Jeffery S [Naperville, IL

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.

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.

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.

6. Vacuum purification room and upper level offices Bureau ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

6. Vacuum purification room and upper level offices - Bureau of Mines Boulder City Experimental Station, Titanium Research Building, Date Street north of U.S. Highway 93, Boulder City, Clark County, NV

Trace impurities analysis determined by neutron activation in the PbI 2 crystal semiconductor

NASA Astrophysics Data System (ADS)

Hamada, M. M.; Oliveira, I. B.; Armelin, M. J.; Mesquita, C. H.

2003-06-01

In this work, a methodology for impurity analysis of PbI 2 was studied to investigate the effectiveness of the purification. Commercial salts were purified by the multi passes zone refining and grown by the Bridgman method. To evaluate the purification efficiency, samples from the bottom, middle and upper sections of the ZR ingot were analyzed after 200, 300 and 500 purification passes, by measurements of the impurity concentrations, using the neutron activation analysis (NAA) technique. There was a significant reduction of the impurities according to the purification numbers. The reduction efficiency was different for each element, namely: Au>Mn>Co˜Ag>K˜Br. The impurity concentration of the crystals grown after 200, 300 and 500 passes and the PbI 2 starting material were analyzed by NAA and plasma optical emission spectroscopy.

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.

Studies on separation and purification of fission (99)Mo from neutron activated uranium aluminum alloy.

PubMed

Rao, Ankita; Kumar Sharma, Abhishek; Kumar, Pradeep; Charyulu, M M; Tomar, B S; Ramakumar, K L

2014-07-01

A new method has been developed for separation and purification of fission (99)Mo from neutron activated uranium-aluminum alloy. Alkali dissolution of the irradiated target (100mg) results in aluminum along with (99)Mo and a few fission products passing into solution, while most of the fission products, activation products and uranium remain undissolved. Subsequent purification steps involve precipitation of aluminum as Al(OH)3, iodine as AgI/AgIO3 and molybdenum as Mo-α-benzoin oxime. Ruthenium is separated by volatilization as RuO4 and final purification of (99)Mo was carried out using anion exchange method. The radiochemical yield of fission (99)Mo was found to be >80% and the purity of the product was in conformity with the international pharmacopoeia standards. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hyperentanglement purification using imperfect spatial entanglement.

PubMed

Wang, Tie-Jun; Mi, Si-Chen; Wang, Chuan

2017-02-06

As the interaction between the photons and the environment which will make the entangled photon pairs in less entangled states or even in mixed states, the security and the efficiency of quantum communication will decrease. We present an efficient hyperentanglement purification protocol that distills nonlocal high-fidelity hyper-entangled Bell states in both polarization and spatial-mode degrees of freedom from ensembles of two-photon system in mixed states using linear optics. Here, we consider the influence of the photon loss in the channel which generally is ignored in the conventional entanglement purification and hyperentanglement purification (HEP) schemes. Compared with previous HEP schemes, our HEP scheme decreases the requirement for nonlocal resources by employing high-dimensional mode-check measurement, and leads to a higher fidelity, especially in the range where the conventional HEP schemes become invalid but our scheme still can work.

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate).

PubMed

Coelho, D F; Silveira, E; Pessoa Junior, A; Tambourgi, E B

2013-02-01

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.

[Purification of arsenic-binding proteins in hamster plasma after oral administration of arsenite].

PubMed

Wang, Wenwen; Zhang, Min; Li, Chunhui; Qin, Yingjie; Hua, Naranmandura

2013-01-01

To purify the arsenic-binding proteins (As-BP) in hamster plasma after a single oral administration of arsenite (iAs(III)). Arsenite was given to hamsters in a single dose. Three types of HPLC columns, size exclusion, gel filtration and anion exchange columns, combined with an inductively coupled argon plasma mass spectrometer (ICP MS) were used to purify the As-BP in hamster plasma. SDS-PAGE was used to confirm the arsenic-binding proteins at each purification step. The three-step purification process successfully separated As-BP from other proteins (ie, arsenic unbound proteins) in hamster plasma. The molecular mass of purified As-BP in plasma was approximately 40-50 kD on SDS-PAGE. The three-step purification method is a simple and fast approach to purify the As-BP in plasma samples.

The Center for Optimized Structural Studies (COSS) platform for automation in cloning, expression, and purification of single proteins and protein-protein complexes.

PubMed

Mlynek, Georg; Lehner, Anita; Neuhold, Jana; Leeb, Sarah; Kostan, Julius; Charnagalov, Alexej; Stolt-Bergner, Peggy; Djinović-Carugo, Kristina; Pinotsis, Nikos

2014-06-01

Expression in Escherichia coli represents the simplest and most cost effective means for the production of recombinant proteins. This is a routine task in structural biology and biochemistry where milligrams of the target protein are required in high purity and monodispersity. To achieve these criteria, the user often needs to screen several constructs in different expression and purification conditions in parallel. We describe a pipeline, implemented in the Center for Optimized Structural Studies, that enables the systematic screening of expression and purification conditions for recombinant proteins and relies on a series of logical decisions. We first use bioinformatics tools to design a series of protein fragments, which we clone in parallel, and subsequently screen in small scale for optimal expression and purification conditions. Based on a scoring system that assesses soluble expression, we then select the top ranking targets for large-scale purification. In the establishment of our pipeline, emphasis was put on streamlining the processes such that it can be easily but not necessarily automatized. In a typical run of about 2 weeks, we are able to prepare and perform small-scale expression screens for 20-100 different constructs followed by large-scale purification of at least 4-6 proteins. The major advantage of our approach is its flexibility, which allows for easy adoption, either partially or entirely, by any average hypothesis driven laboratory in a manual or robot-assisted manner.

New ligation independent cloning vectors for expression of recombinant proteins with a self-cleaving CPD/6xHis-tag.

PubMed

Biancucci, Marco; Dolores, Jazel S; Wong, Jennifer; Grimshaw, Sarah; Anderson, Wayne F; Satchell, Karla J F; Kwon, Keehwan

2017-01-05

Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.

Development, upscaling and validation of the purification process for human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII produced in a human cell-line.

PubMed

Winge, Stefan; Yderland, Louise; Kannicht, Christoph; Hermans, Pim; Adema, Simon; Schmidt, Torben; Gilljam, Gustav; Linhult, Martin; Tiemeyer, Maya; Belyanskaya, Larisa; Walter, Olaf

2015-11-01

Human-cl rhFVIII (Nuwiq®), a new generation recombinant factor VIII (rFVIII), is the first rFVIII produced in a human cell-line approved by the European Medicines Agency. To describe the development, upscaling and process validation for industrial-scale human-cl rhFVIII purification. The purification process involves one centrifugation, two filtration, five chromatography columns and two dedicated pathogen clearance steps (solvent/detergent treatment and 20 nm nanofiltration). The key purification step uses an affinity resin (VIIISelect) with high specificity for FVIII, removing essentially all host-cell proteins with >80% product recovery. The production-scale multi-step purification process efficiently removes process- and product-related impurities and results in a high-purity rhFVIII product, with an overall yield of ∼50%. Specific activity of the final product was >9000 IU/mg, and the ratio between active FVIII and total FVIII protein present was >0.9. The entire production process is free of animal-derived products. Leaching of potential harmful compounds from chromatography resins and all pathogens tested were below the limit of quantification in the final product. Human-cl rhFVIII can be produced at 500 L bioreactor scale, maintaining high purity and recoveries. The innovative purification process ensures a high-purity and high-quality human-cl rhFVIII product with a high pathogen safety margin. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Purification of lanthanides for double beta decay experiments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Polischuk, O. G.; Barabash, A. S.; Belli, P.

2013-08-08

There are several potentially double beta active isotopes among the lanthanide elements. However, even high purity grade lanthanide compounds contain {sup 238}U, {sup 226}Ra and {sup 232,228}Th typically on the level of ∼ (0.1 - 1) Bq/kg. The liquid-liquid extraction technique was used to remove traces of U, Ra and Th from CeO{sub 2}, Nd{sub 2}O{sub 3} and Gd{sub 2}O{sub 3}. The radioactive contamination of the samples before and after the purification was tested by using ultra-low-background HPGe γ spectrometry at the underground Gran Sasso National Laboratories of the INFN (Italy). After the purification the radioactive contamination of gadolinium oxidemore » by Ra and Th was decreased at least one order of magnitude. The efficiency of the approach to purify cerium oxide from Ra was on same level, while the radioactive contamination of neodymium sample before and after the purification is below the sensitivity of analytical methods. The purification method is much less efficient for chemically very similar radioactive elements like lanthanum, lutetium and actinium. R and D of the methods to remove the pollutions with improved efficiency is in progress.« less

Porous graphene-based membranes for water purification from metal ions at low differential pressures.

PubMed

Park, Jaewoo; Bazylewski, Paul; Fanchini, Giovanni

2016-05-14

A new generation of membranes for water purification based on weakly oxidized and nanoporous few-layer graphene is here introduced. These membranes dramatically decrease the high energy requirements of water purification by reverse osmosis. They combine the advantages of porous and non-oxidized single-layer graphene, offering energy-efficient water filtration at relatively low differential pressures, and highly oxidized graphene oxide, exhibiting high performance in terms of impurity adsorption. In the reported fabrication process, leaks between juxtaposed few-layer graphene flakes are sealed by thermally annealed colloidal silica, in a treatment that precedes the opening of (sub)nanometre-size pores in graphene. This process, explored for the first time in this work, results in nanoporous graphene flakes that are water-tight at the edges without occluding the (sub)nanopores. With this method, removal of impurities from water occurs through a combination of size-based pore rejection and pore-edge adsorption. Thinness of graphene flakes allows these membranes to achieve water purification from metal ions in concentrations of few parts-per-million at differential pressures as low as 30 kPa, outperforming existing graphene or graphene oxide purification systems with comparable flow rates.

Porous graphene-based membranes for water purification from metal ions at low differential pressures

NASA Astrophysics Data System (ADS)

Park, Jaewoo; Bazylewski, Paul; Fanchini, Giovanni

2016-05-01

A new generation of membranes for water purification based on weakly oxidized and nanoporous few-layer graphene is here introduced. These membranes dramatically decrease the high energy requirements of water purification by reverse osmosis. They combine the advantages of porous and non-oxidized single-layer graphene, offering energy-efficient water filtration at relatively low differential pressures, and highly oxidized graphene oxide, exhibiting high performance in terms of impurity adsorption. In the reported fabrication process, leaks between juxtaposed few-layer graphene flakes are sealed by thermally annealed colloidal silica, in a treatment that precedes the opening of (sub)nanometre-size pores in graphene. This process, explored for the first time in this work, results in nanoporous graphene flakes that are water-tight at the edges without occluding the (sub)nanopores. With this method, removal of impurities from water occurs through a combination of size-based pore rejection and pore-edge adsorption. Thinness of graphene flakes allows these membranes to achieve water purification from metal ions in concentrations of few parts-per-million at differential pressures as low as 30 kPa, outperforming existing graphene or graphene oxide purification systems with comparable flow rates.

Purification effect of two typical water source vegetation buffer zones on land-sourced pollutants

NASA Astrophysics Data System (ADS)

Li, Gang

2017-03-01

Two vegetation buffer zones (tree-shrub-grass pattern and tree-grass pattern) were selected as test objects around Siming reservoir in Yuyao City of China. The effect of the storm runoff intensity (low and high intensity) and the buffer zone width (1 m, 3 m, 5 m, 7 m, 9 m, 12 m, 16 m) on pollutants (suspended solids, ammonium nitrogen and total phosphorus) was studied by the artificial simulation runoff. The results showed that with the increase of the width of buffer zone, the pollutant concentration was decreased. The purification effect of the two buffer zones on suspended solids and total phosphorus was basically stable at 52-55% and 34-37%, respectively. But the purification effect on ammonium nitrogen was the tree-shrub-grass pattern (69.7%) significantly better than that of tree-grass pattern (52.1%). The purification rate at the low runoff intensity was 1.8-2.0 times that at the high runoff intensity. The relationship between the purification rate and buffer zone width can be expressed by the natural logarithm equation, and the model adjustment coefficient was greater than 0.92.

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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

[Selection and purification potential evaluation of woody plant in vertical flow constructed wetlands in the subtropical area].

PubMed

Chen, Yong-Hua; Wu, Xiao-Fu; Hao, Jun; Chen, Ming-Li; Zhu, Guang-Yu

2014-02-01

In order to solve the problem that wetland herbaceous plants tend to die during winter in subtropics areas, selection and purification potential evaluation experiments were carried out by introducing into the constructed wetlands 16 species of woody wetland plants. Cluster analysis was performed by including the morphological characteristics, physiological characteristics, as well as nitrogen and phosphorus accumulation of the woody wetland plants. The results indicated that there were significant differences among the tested woody plants in their survival rate, height increase, root length increase and vigor, Chlorophyll content, Superoxide dismutase, Malonaldehyde, Proline, Peroxidase, biomass, average concentration and accumulation of nitrogen and phosphorus. Based on the established evaluation system, the tested plants were clustered into 3 groups. The plants in the 1st group possessing high purification potentials are Nerium oleander and Hibiscus syriacus. Those in the 2nd group possessing moderate purification potentials are Trachycarpus fortune, Llex latifolia Thunb., Gardenia jasminoides, Serissa foetida and Ilex crenatacv Convexa. And those in the 3rd group with low purification potentials are Jasminum udiflorum, Hedera helix, Ligustrum vicaryi, Ligustrum lucidum, Buxus sempervives, Murraya paniculata, Osmanthus fragrans, Mahoniafortune and Photinia serrulata.

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

Guidelines to reach high-quality purified recombinant proteins.

PubMed

Oliveira, Carla; Domingues, Lucília

2018-01-01

The final goal in recombinant protein production is to obtain high-quality pure protein samples. Indeed, the successful downstream application of a recombinant protein depends on its quality. Besides production, which is conditioned by the host, the quality of a recombinant protein product relies mainly on the purification procedure. Thus, the purification strategy must be carefully designed from the molecular level. On the other hand, the quality control of a protein sample must be performed to ensure its purity, homogeneity and structural conformity, in order to validate the recombinant production and purification process. Therefore, this review aims at providing succinct information on the rational purification design of recombinant proteins produced in Escherichia coli, specifically the tagging purification, as well as on accessible tools for evaluating and optimizing protein quality. The classical techniques for structural protein characterization-denaturing protein gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), dynamic light scattering (DLS) and circular dichroism (CD)-are revisited with focus on the protein and their main advantages and disadvantages. Furthermore, methods for determining protein concentration and protein storage are also presented. The guidelines compiled herein will aid preparing pure, soluble and homogeneous functional recombinant proteins from the very beginning of the molecular cloning design.

An expression vector tailored for large-scale, high-throughput purification of recombinant proteins ☆

PubMed Central

Donnelly, Mark I.; Zhou, Min; Millard, Cynthia Sanville; Clancy, Shonda; Stols, Lucy; Eschenfeldt, William H.; Collart, Frank R.; Joachimiak, Andrzej

2009-01-01

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his6-tag–maltose-binding protein (MBP), intended to facilitate purification and enhance proteins’ solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his6-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his6-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his6-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his6-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his6-tag. PMID:16497515

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.

Development of RAP Tag, a Novel Tagging System for Protein Detection and Purification.

PubMed

Fujii, Yuki; Kaneko, Mika K; Ogasawara, Satoshi; Yamada, Shinji; Yanaka, Miyuki; Nakamura, Takuro; Saidoh, Noriko; Yoshida, Kanae; Honma, Ryusuke; Kato, Yukinari

2017-04-01

Affinity tag systems, possessing high affinity and specificity, are useful for protein detection and purification. The most suitable tag for a particular purpose should be selected from many available affinity tag systems. In this study, we developed a novel affinity tag called the "RAP tag" system, which comprises a mouse antirat podoplanin monoclonal antibody (clone PMab-2) and the RAP tag (DMVNPGLEDRIE). This system is useful not only for protein detection in Western blotting, flow cytometry, and sandwich enzyme-linked immunosorbent assay, but also for protein purification.

Isolation and purification of antigenic components of Cryptococcus.

PubMed

Wozniak, Karen L; Levitz, Stuart M

2009-01-01

The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species.

[Development of new magnetic bead separation and purification instrument].

PubMed

Xu, Yingyuan; Chen, Yi

2014-05-01

The article describes the development of new magnetic bead separation and purification instrument. The main application of the instrument is to capture tubercle bacillus from sputum. It is a pretreatment instrument and provides a new platform to help doctors to diagnose bacillary phthisis. Not only could it be used for tubercle bacillus capturing, but also for gene, protein and cell separating and purification. Because the controller of the instrument is 16-bit single chip microcomputer, the cost could be greatly reduced and it will be widely used in China.

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

Studying breaking of inverted emulsions with thermolysis purification TD600

NASA Astrophysics Data System (ADS)

Tarasova, G. I.; Shevaga, O. N.; Grachyova, E. O.

2018-03-01

Currently, emulsions are used in many branches of industry and agriculture. It explains significant attention paid to issues in production, stabilization and breaking of emulsion. Besides, producing steady emulsions is of importance in many processes; the reverse problem, that of demulsification, is important as well in oil production and treatment of oil emulsion waste water. This paper studies the breaking (demulsification) of inverted emulsions with the help of thermolysis purification TD600, produced by thermal modification of purification, a large-scale waste of the sugar industry.

Expression and purification of recombinant nattokinase in Spodoptera frugiperda cells.

PubMed

Li, Xiaoxiang; Wang, Xiaoli; Xiong, Shaoling; Zhang, Jing; Cai, Litao; Yang, Yanyan

2007-10-01

A recombinant baculovirus, rv-egfp-NK, containing a reporter gene encoding the enhanced green fluorescent protein (EGFP), was used to express nattokinase (NK), a fibrinolytic enzyme, in Spodoptera frugiperda (SF-9) cells. The recombinant protein also included a histidine tag for purification using Ni(2+) resins. The recombinant NK, approximately 30 kDa, retained fibrinolytic activity (60 U/ml). The integration of the EGFP expression cassette in the Bac-to-Bac system is thus an effective method for the expression and purification of recombinant NK protein in Spodoptera frugiperda insect cells.

Addressing the medicinal chemistry bottleneck: a lean approach to centralized purification.

PubMed

Weller, Harold N; Nirschl, David S; Paulson, James L; Hoffman, Steven L; Bullock, William H

2012-09-10

The use of standardized lean manufacturing principles to improve drug discovery productivity is often thought to be at odds with fostering innovation. This manuscript describes how selective implementation of a lean optimized process, in this case centralized purification for medicinal chemistry, can improve operational productivity and increase scientist time available for innovation. A description of the centralized purification process is provided along with both operational and impact (productivity) metrics, which indicate lower cost, higher output, and presumably more free time for innovation as a result of the process changes described.

Synthesis of capped RNA using a DMT group as a purification handle.

PubMed

Veliath, Elizabeth; Gaffney, Barbara L; Jones, Roger A

2014-01-01

We report a new method for synthesis of capped RNA or 2'-OMe RNA that uses a N(2-)4,4'-dimethoxytrityl (DMT) group as a lipophilic purification handle to allow convenient isolation and purification of the capped RNA. The DMT group is easily removed under mild conditions without degradation of the cap. We have used this approach to prepare capped 10- and 20-mers. This method is compatible with the many condensation reactions that have been reported for preparation of capped RNA or cap analogues.

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 development other of HIC-compatible protein purification schemes. PMID:25254496

Jeffrey Blackburn | NREL

Science.gov Websites

-dimensional carbon and includes the synthesis, purification, separation, and characterization of single-walled conversion Synthesis, purification, separation, and characterization of single-walled carbon nanotubes Synthesis, characterization, and device integration of graphen Hydrogen storage Photovoltaic materials and

Carbon nanotube membranes with ultrahigh specific adsorption capacity for water desalination and purification.

PubMed

Yang, Hui Ying; Han, Zhao Jun; Yu, Siu Fung; Pey, Kin Leong; Ostrikov, Kostya; Karnik, Rohit

2013-01-01

Development of technologies for water desalination and purification is critical to meet the global challenges of insufficient water supply and inadequate sanitation, especially for point-of-use applications. Conventional desalination methods are energy and operationally intensive, whereas adsorption-based techniques are simple and easy to use for point-of-use water purification, yet their capacity to remove salts is limited. Here we report that plasma-modified ultralong carbon nanotubes exhibit ultrahigh specific adsorption capacity for salt (exceeding 400% by weight) that is two orders of magnitude higher than that found in the current state-of-the-art activated carbon-based water treatment systems. We exploit this adsorption capacity in ultralong carbon nanotube-based membranes that can remove salt, as well as organic and metal contaminants. These ultralong carbon nanotube-based membranes may lead to next-generation rechargeable, point-of-use potable water purification appliances with superior desalination, disinfection and filtration properties.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Feng Xie; Hong Li; Jianzhu Cao

A reform will be implemented in the helium purification system of the 10 MW High Temperature Gas-cooled Test Reactor (HTR-10) in China. The measurement of the gamma dose rates of facilities, including valves, pipes, dust filter, etc., in the purification system of the HTR-10, has been performed. The results indicated that most radiation nuclides are concentrated in the dust filter and facilities at the entrance of the helium purification system upstream of the dust filter. Other facilities have the same gamma dose rate level as the background. Based on the previous study and experiences in AVR, the measurement results canmore » be understood that the radioactive dust carried by the helium gas was filtered by the dust filter. It provides important insights for the decontamination and decommissioning of facilities in the primary loop, especially in the helium purification system of the HTR-10 as well as the High Temperature Reactor-Pebble bed Modules (HTR-PM). (authors)« less

Rapid purification of diastereoisomers from Piper kadsura using supercritical fluid chromatography with chiral stationary phases.

PubMed

Xin, Huaxia; Dai, Zhuoshun; Cai, Jianfeng; Ke, Yanxiong; Shi, Hui; Fu, Qing; Jin, Yu; Liang, Xinmiao

2017-08-04

Supercritical fluid chromatography (SFC) with chiral stationary phases (CSPs) is an advanced solution for the separation of achiral compounds in Piper kadsura. Analogues and stereoisomers are abundant in natural products, but there are obstacles in separation using conventional method. In this paper, four lignan diastereoisomers, (-)-Galbelgin, (-)-Ganschisandrin, Galgravin and (-)-Veraguensin, from Piper kadsura were separated and purified by chiral SFC. Purification strategy was designed, considering of the compound enrichment, sample purity and purification throughput. Two-step achiral purification method on chiral preparative columns with stacked automated injections was developed. Unconventional mobile phase modifier dichloromethane (DCM) was applied to improve the sample solubility. Four diastereoisomers was prepared at the respective weight of 103.1mg, 10.0mg, 152.3mg and 178.6mg from 710mg extract with the purity of greater than 98%. Copyright © 2017 Elsevier B.V. All rights reserved.

Isolation, purification, and partial characterization of a membrane-bound Cl-/HCO3--activated ATPase complex from rat brain with sensitivity to GABAAergic ligands.

PubMed

Menzikov, Sergey A

2017-02-07

This study describes the isolation and purification of a protein complex with [Formula: see text]-ATPase activity and sensitivity to GABA A ergic ligands from rat brain plasma membranes. The ATPase complex was enriched using size-exclusion, affinity, and ion-exchange chromatography. The fractions obtained at each purification step were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), which revealed four subunits with molecular mass ∼48, 52, 56, and 59 kDa; these were retained at all stages of the purification process. Autoradiography revealed that the ∼52 and 56 kDa subunits could bind [ 3 H]muscimol. The [Formula: see text]-ATPase activity of this enriched protein complex was regulated by GABA A ergic ligands but was not sensitive to blockers of the NKCC or KCC cotransporters.

Carbon-11 choline: synthesis, purification, and brain uptake inhibition by 2-dimethylaminoethanol

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rosen, M.A.; Jones, R.M.; Yano, Y.

We report an improved method for the synthesis and purification of (11C)methylcholine from the precursors (11C)methyliodide and 2-dimethylaminoethanol (deanol). Preparation time, including purification, is 35 min postbombardment. Forty millicuries of purified injectable (11C)choline were produced with a measured specific activity of greater than 300 Ci/mmol and a radiochemical purity greater than 98%. The decay corrected radiochemical yield for the synthesis and purification was approximately 50%. Residual precursor deanol, which inhibits brain uptake of choline, is removed by a rapid preparative high performance liquid chromatography (HPLC) method using a reverse phase cyano column with a biologically compatible 100% water eluent. Evaporationmore » alone did not completely remove the deanol precursor. Brain uptake of the (11C)choline product was six times greater after HPLC removal of deanol because doses of less than 1 microgram/kg significantly inhibit (14C)choline brain uptake.« less

Multi-copy entanglement purification with practical spontaneous parametric down conversion sources

NASA Astrophysics Data System (ADS)

Zhang, Shuai-Shuai; Shu, Qi; Zhou, Lan; Sheng, Yu-Bo

2017-06-01

Entanglement purification is to distill the high quality entanglement from the low quality entanglement with local operations and classical communications. It is one of the key technologies in long-distance quantum communication. We discuss an entanglement purification protocol (EPP) with spontaneous parametric down conversion (SPDC) sources, in contrast to previous EPP with multi-copy mixed states, which requires ideal entanglement sources. We show that the SPDC source is not an obstacle for purification, but can benefit the fidelity of the purified mixed state. This EPP works for linear optics and is feasible in current experiment technology. Project supported by the National Natural Science Foundation of China (Grant Nos. 11474168 and 61401222), the Natural Science Foundation of Jiangsu Province, China (Grant No. BK20151502), the Qing Lan Project in Jiangsu Province, China, and a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, China.

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.

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.

Purification of Nanoscale Electron-Beam-Induced Platinum Deposits via a Pulsed Laser-Induced Oxidation Reaction

DOE PAGES

Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon; ...

2014-11-05

Platinum–carbon deposits made via electron-beam-induced deposition were purified in this study via a pulsed laser-induced oxidation reaction and erosion of the amorphous carbon to form pure platinum. Purification proceeds from the top down and is likely catalytically facilitated via the evolving platinum layer. Thermal simulations suggest a temperature threshold of ~485 K, and the purification rate is a function of the PtC 5 thickness (80–360 nm) and laser pulse width (1–100 μs) in the ranges studied. The thickness dependence is attributed to the ~235 nm penetration depth of the PtC 5 composite at the laser wavelength, and the pulse-width dependencemore » is attributed to the increased temperatures achieved at longer pulse widths. Finally, remarkably fast purification is realized at cumulative laser exposure times of less than 1 s.« less

Purification of Nanoscale Electron-Beam-Induced Platinum Deposits via a Pulsed Laser-Induced Oxidation Reaction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stanford, Michael G.; Lewis, Brett B.; Noh, Joo Hyon

Platinum–carbon deposits made via electron-beam-induced deposition were purified in this study via a pulsed laser-induced oxidation reaction and erosion of the amorphous carbon to form pure platinum. Purification proceeds from the top down and is likely catalytically facilitated via the evolving platinum layer. Thermal simulations suggest a temperature threshold of ~485 K, and the purification rate is a function of the PtC 5 thickness (80–360 nm) and laser pulse width (1–100 μs) in the ranges studied. The thickness dependence is attributed to the ~235 nm penetration depth of the PtC 5 composite at the laser wavelength, and the pulse-width dependencemore » is attributed to the increased temperatures achieved at longer pulse widths. Finally, remarkably fast purification is realized at cumulative laser exposure times of less than 1 s.« less

Antimicrobial Peptide Production and Purification.

PubMed

Suda, Srinivas; Field, Des; Barron, Niall

2017-01-01

Antimicrobial peptides (AMPs) are natural defense compounds which are synthesized as ribosomal gene-encoded pre-peptides and produced by all living organisms. AMPs are small peptides, usually cationic and typically have hydrophobic residues which interact with cell membranes and have either a narrow or broad spectrum of biological activity. AMPs are isolated from the natural host or heterologously expressed in other hosts such as Escherichia coli. The proto-typical lantibiotic Nisin is a widely used AMP that is produced by the food-grade organism Lactococcus lactis. Although AMP production and purification procedures require optimization for individual AMPs, the Nisin production and purification protocol outlined in this chapter can be easily applied with minor modifications for the production and purification of other lantibiotics or AMPs. While Nisin is produced and secreted into the supernatant, steps to recover Nisin from both cell-free supernatant and cell pellet are outlined in detail.

Monolith-based immobilized metal affinity chromatography increases production efficiency for plasmid DNA purification.

PubMed

Shin, Min Jae; Tan, Lihan; Jeong, Min Ho; Kim, Ji-Heung; Choe, Woo-Seok

2011-08-05

Immobilized metal affinity monolith column as a new class of chromatographic support is shown to be superior to conventional particle-based column as plasmid DNA (pDNA) purification platform. By harnessing the affinity of endotoxin to copper ions in the solution, a majority of endotoxin (90%) was removed from the alkaline cell lysate using CuCl(2)-induced precipitation. RNA and remaining endotoxin were subsequently removed to below detection limit with minimal loss of pDNA using either monolith or particle-based column. Monolith column has the additional advantage of feed concentration and flowrate-independent dynamic binding capacity for RNA molecules, enabling purification process to be conducted at high feed RNA concentration and flowrate. The use of monolith column gives three fold increased productivity of pDNA as compared to particle-based column, providing a more rapid and economical platform for pDNA purification. Copyright © 2011 Elsevier B.V. All rights reserved.

The SNO+ Scintillator Purification Plant and Projected Sensitivity to Solar Neutrinos in the Pure Scintillator Phase

NASA Astrophysics Data System (ADS)

Pershing, Teal; SNO+ Collaboration

2016-03-01

The SNO+ detector is a neutrino and neutrinoless double-beta decay experiment utilizing the renovated SNO detector. In the second phase of operation, the SNO+ detector will contain 780 tons of organic liquid scintillator composed of 2 g/L 2,5-diphenyloxazole (PPO) in linear alkylbenzene (LAB). In this phase, SNO+ will strive to detect solar neutrinos in the sub-MeV range, including CNO production neutrinos and pp production neutrinos. To achieve the necessary detector sensitivity, a four-part scintillator purification plant has been constructed in SNOLAB for the removal of ionic and radioactive impurities. We present an overview of the SNO+ scintillator purification plant stages, including distillation, water extraction, gas stripping, and metal scavenger columns. We also give the projected SNO+ sensitivities to various solar-produced neutrinos based on the scintillator plant's projected purification efficiency.

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed

Tweeten, K A; Bulla, L A; Consigli, R A

1977-09-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

PubMed Central

Tweeten, K A; Bulla, L A; Consigli, R A

1977-01-01

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid. Images PMID:334076

Novel cross-linked alcohol-insoluble solid (CL-AIS) affinity gel from pea pod for pectinesterase purification.

PubMed

Wu, Ming-Chang; Lin, Guan-Hui; Wang, Yuh-Tai; Jiang, Chii-Ming; Chang, Hung-Min

2005-10-05

Alcohol-insoluble solids (AIS) from pea pod were cross-linked (CL-AIS) and used as an affinity gel matrix to isolate pectin esterases (PEs) from tendril shoots of chayote (TSC) and jelly fig achenes (JFA), and the results were compared with those isolated by ion-exchange chromatography with a commercial resin. CL-AIS gel matrix in a column displayed poor absorption and purification fold of PE; however, highly methoxylated CL-AIS (HM-CL-AIS), by exposing CL-AIS to methanolic sulfuric acid to increase the degree of esterification (DE) to 92%, facilitated the enzyme purification. The purified TSC PE and JFA PE by the HM-CL-AIS column were proofed as a single band on an SDS-PAGE gel, showing that the HM-CL-AIS column was a good matrix for purification of PE, either with alkaline isoelectric point (pI) (TSC PE) or with acidic pI (JFA PE).

Magnetic particles as powerful purification tool for high sensitive mass spectrometric screening procedures.

PubMed

Peter, Jochen F; Otto, Angela M

2010-02-01

The effective isolation and purification of proteins from biological fluids is the most crucial step for a successful protein analysis when only minute amounts are available. While conventional purification methods such as dialysis, ultrafiltration or protein precipitation often lead to a marked loss of protein, SPE with small-sized particles is a powerful alternative. The implementation of particles with superparamagnetic cores facilitates the handling of those particles and allows the application of particles in the nanometer to low micrometer range. Due to the small diameters, magnetic particles are advantageous for increasing sensitivity when using subsequent MS analysis or gel electrophoresis. In the last years, different types of magnetic particles were developed for specific protein purification purposes followed by analysis or screening procedures using MS or SDS gel electrophoresis. In this review, the use of magnetic particles for different applications, such as, the extraction and analysis of DNA/RNA, peptides and proteins, is described.

Preparative isolation and purification of seven isoflavones from Belamcanda chinensis.

PubMed

Lee, Yeon Sil; Kim, Seon Ha; Kim, Jin Kyu; Lee, Sanghyun; Jung, Sang Hoon; Lim, Soon Sung

2011-01-01

Isoflavonoids from Belamcanda chinensis are known to have a number of physiological benefits including anti-inflammatory, anti-angiogenic and anti-mutagenic properties. However, there have been no reports on the effective isolation and purification of isoflavonoids from B. chinensis. To develop an efficient method for the preparative isolation and purification of isoflavones from B. chinensis by high-speed counter-current chromatography (HSCCC). A two-step HSCCC isolation method was developed using solvent system of n-hexane-ethyl acetate-2-propanol-methanol-water (5:6:2:3.5:6, v/v) and of ethyl acetate-methanol-water (10:2:9, v/v). FLASH purification system (45% methanol, isocratic) was also used for further purification. The purities and chemical structures of the isolated compounds were determined by high-performance liquid chromatography-photodiode array detection (HPLC-PDA), electrospray ionisation-mass spectrometry (ESI-MS), ¹H- and ¹³C-nuclear magnetic resonance spectrometry (NMR) and nuclear overhauser enhancement (NOE). HSCCC was successfully used for the preparative separation and purification of seven isoflavones, including tectoridin (145.4 mg, 97.5%), iridin (77.9 mg, 94.0%), irilin D (42.0 mg, 92.0%), tectorigenin (294.1 mg, 98.6%), iristectorigenin A (86.8 mg, 93.4%), irigenin (141.8 mg, 95.8%) and irisflorentin (73.4 mg, 94.7%) from the rhizomes of B. chinensis. Two isoflavone glycosides and five isoflavone derivatives were successfully isolated and purified from the crude methanol extract of dried rhizomes of the B. chinensis by HSCCC. Copyright © 2011 John Wiley & Sons, Ltd.

[Alcohol-purification technology and its particle sedimentation process in manufactory of Fufang Kushen injection].

PubMed

Liu, Xiaoqian; Tong, Yan; Wang, Jinyu; Wang, Ruizhen; Zhang, Yanxia; Wang, Zhimin

2011-11-01

Fufang Kushen injection was selected as the model drug, to optimize its alcohol-purification process and understand the characteristics of particle sedimentation process, and to investigate the feasibility of using process analytical technology (PAT) on traditional Chinese medicine (TCM) manufacturing. Total alkaloids (calculated by matrine, oxymatrine, sophoridine and oxysophoridine) and macrozamin were selected as quality evaluation markers to optimize the process of Fufang Kushen injection purification with alcohol. Process parameters of particulate formed in the alcohol-purification, such as the number, density and sedimentation velocity, were also determined to define the sedimentation time and well understand the process. The purification process was optimized as that alcohol is added to the concentrated extract solution (drug material) to certain concentration for 2 times and deposited the alcohol-solution containing drug-material to sediment for some time, i.e. 60% alcohol deposited for 36 hours, filter and then 80% -90% alcohol deposited for 6 hours in turn. The content of total alkaloids was decreased a little during the depositing process. The average settling time of particles with the diameters of 10, 25 microm were 157.7, 25.2 h in the first alcohol-purified process, and 84.2, 13.5 h in the second alcohol-purified process, respectively. The optimized alcohol-purification process remains the marker compositions better and compared with the initial process, it's time saving and much economy. The manufacturing quality of TCM-injection can be controlled by process. PAT pattern must be designed under the well understanding of process of TCM production.

Use of anionic denaturing detergents to purify insoluble proteins after overexpression

PubMed Central

2012-01-01

Background Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. Results Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. Conclusion Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology. PMID:23231964

The Partial Purification and Characterization of Lactate Dehydrogenase.

ERIC Educational Resources Information Center

Wolf, Edward C.

1988-01-01

Offers several advantages over other possibilities as the enzyme of choice for a student's first exposure to a purification scheme. Uses equipment and materials normally found in biochemistry laboratories. Incorporates several important biochemical techniques including spectrophotometry, chromatography, centrifugation, and electrophoresis. (MVL)

Isolation and Purification of Antigenic Components of Cryptococcus

PubMed Central

Wozniak, Karen L.; Levitz, Stuart M.

2012-01-01

The encapsulated fungal pathogens Cryptococcus neoformans and Cryptococcus gattii are significant agents of life-threatening infections, particularly in persons with suppressed cell-mediated immunity. This chapter provides detailed methodology for the purification of two of the major antigen fractions of C. neoformans: glucuronoxylomannan (GXM) and mannoprotein (MP). GXM is the primary component of the polysaccharide capsule, which is the major cryptococcal virulence factor. In contrast, MPs have been identified as key antigens that stimulate T-cell responses. Purification of GXM and MP should assist investigators studying the antigenic, biochemical, and virulence properties of Cryptococcus species. PMID:19089377

Magnetic techniques for the isolation and purification of proteins and peptides

PubMed Central

Safarik, Ivo; Safarikova, Mirka

2004-01-01

Isolation and separation of specific molecules is used in almost all areas of biosciences and biotechnology. Diverse procedures can be used to achieve this goal. Recently, increased attention has been paid to the development and application of magnetic separation techniques, which employ small magnetic particles. The purpose of this review paper is to summarize various methodologies, strategies and materials which can be used for the isolation and purification of target proteins and peptides with the help of magnetic field. An extensive list of realised purification procedures documents the efficiency of magnetic separation techniques. PMID:15566570

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

PubMed

Genji, Takahisa; Nozawa, Akira; Tozawa, Yuzuru

2010-10-01

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield. Copyright © 2010 Elsevier Inc. All rights reserved.

Combustion water purification techniques influence on OBT analysing using liquid scintillation counting method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Varlam, C.; Vagner, I.; Faurescu, I.

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 themore » 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.« less

Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

PubMed Central

Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G.

2017-01-01

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity (Kd ∼ 10−14–10−17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays. PMID:28607052

Experimental studies on islets isolation, purification and function in rats

PubMed Central

Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

2015-01-01

To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient. PMID:26885021

Effects of L-arginine on solubilization and purification of plant membrane proteins.

PubMed

Arakawa, Junji; Uegaki, Masamichi; Ishimizu, Takeshi

2011-11-01

Biochemical analysis of membrane proteins is problematic at the level of solubilization and/or purification because of their hydrophobic nature. Here, we developed methods for efficient solubilization and purification of membrane proteins using L-arginine. The addition of 100 mM of basic amino acids (L-arginine, L-lysine, and L-ornithine) to a detergent-containing solubilization buffer enhanced solubilization (by 2.6-4.3 fold) of a model membrane protein-polygalacturonic acid synthase. Of all the amino acids, arginine was the most effective additive for solubilization of this membrane protein. Arginine addition also resulted in the best solubilization of other plant membrane proteins. Next, we examined the effects of arginine on purification of a model membrane protein. In anion-exchange chromatography, the addition of arginine to the loading and elution buffers resulted in a greater recovery of a membrane protein. In ultrafiltration, the addition of arginine to a protein solution significantly improved the recovery of a membrane protein. These results were thought to be due to the properties of arginine that prevent aggregation of hydrophobic proteins. Taken together, the results of our study showed that arginine is useful for solubilization and purification of aggregate-prone membrane proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Simplified Large-Scale Refolding, Purification, and Characterization of Recombinant Human Granulocyte-Colony Stimulating Factor in Escherichia coli

PubMed Central

Kim, Chang Kyu; Lee, Chi Ho; Lee, Seung-Bae; Oh, Jae-Wook

2013-01-01

Granulocyte-colony stimulating factor (G-CSF) is a pleiotropic cytokine that stimulates the development of committed hematopoietic progenitor cells and enhances the functional activity of mature cells. Here, we report a simplified method for fed-batch culture as well as the purification of recombinant human (rh) G-CSF. The new system for rhG-CSF purification was performed using not only temperature shift strategy without isopropyl-l-thio-β-d-galactoside (IPTG) induction but also the purification method by a single step of prep-HPLC after the pH precipitation of the refolded samples. Through these processes, the final cell density and overall yield of homogenous rhG-CSF were obtained 42.8 g as dry cell weights, 1.75 g as purified active proteins, from 1 L culture broth, respectively. The purity of rhG-CSF was finally 99% since the isoforms of rhG-CSF could be separated through the prep-HPLC step. The result of biological activity indicated that purified rhG-CSF has a similar profile to the World Health Organization (WHO) 2nd International Standard for G-CSF. Taken together, our results demonstrate that the simple purification through a single step of prep-HPLC may be valuable for the industrial-scale production of biologically active proteins. PMID:24224041

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

Improved purification of immunoglobulin G from plasma by mixed-mode chromatography.

PubMed

Chai, Dong-Sheng; Sun, Yan; Wang, Xiao-Ning; Shi, Qing-Hong

2014-12-01

Efficient loading of immunoglobulin G in mixed-mode chromatography is often a serious bottleneck in the chromatographic purification of immunoglobulin G. In this work, a mixed-mode ligand, 4-(1H-imidazol-1-yl) aniline, was coupled to Sepharose Fast Flow to fabricate AN SepFF adsorbents with ligand densities of 15-64 mmol/L, and the chromatographic performances of these adsorbents were thoroughly investigated to identify a feasible approach to improve immunoglobulin G purification. The results indicate that a critical ligand density exists for immunoglobulin G on the AN SepFF adsorbents. Above the critical ligand density, the adsorbents showed superior selectivity to immunoglobulin G at high salt concentrations, and also exhibited much higher dynamic binding capacities. For immunoglobulin G purification, both the yield and binding capacity increased with adsorbent ligand density along with a decrease in purity. It is difficult to improve the binding capacity, purity, and yield of immunoglobulin G simultaneously in AN SepFF chromatography. By using tandem AN SepFF chromatography, a threefold increase in binding capacity as well as high purity and yield of immunoglobulin G were achieved. Therefore, the tandem chromatography demonstrates that AN SepFF adsorbent is a practical and feasible alternative to MEP HyperCel adsorbents for immunoglobulin G purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Purification and characterization of a trehalase-invertase enzyme with dual activity from Candida utilis.

PubMed

Lahiri, Sagar; Basu, Arghya; Sengupta, Shinjinee; Banerjee, Shakri; Dutta, Trina; Soren, Dhananjay; Chattopadhyay, Krishnananda; Ghosh, Anil K

2012-06-15

Trehalose and sucrose, two important anti-stress non-reducing natural disaccharides, are catabolized by two enzymes, namely trehalase and invertase respectively. In this study, a 175 kDa enzyme protein active against both substrates was purified from wild type Candida utilis and characterized in detail. Substrate specificity assay and activity staining revealed the enzyme to be specific for both sucrose and trehalose. The ratio between trehalase and invertase activity was found to be constant at 1:3.5 throughout the entire study. Almost 40-fold purification and 30% yield for both activities were achieved at the final step of purification. The presence of common enzyme inhibitors, thermal and pH stress had analogous effects on its trehalase and invertase activity. Km values for two activities were similar while Vmax and Kcat also differed by a factor of 3.5. Competition plot for both substrates revealed the two activities to be occurring at the single active site. N-terminal sequencing and MALDI-TOF data analysis revealed higher similarity of the purified protein to previously known neutral trehalases. While earlier workers mentioned independent purification of neutral trehalase or invertase from different sources, the present study reports the purification of a single protein showing dual activity. Copyright © 2012 Elsevier Inc. All rights reserved.

Case studies on the physical-chemical parameters' variation during three different purification approaches destined to treat wastewaters from food industry.

PubMed

Ghimpusan, Marieta; Nechifor, Gheorghe; Nechifor, Aurelia-Cristina; Dima, Stefan-Ovidiu; Passeri, Piero

2017-12-01

The paper presents a set of three interconnected case studies on the depuration of food processing wastewaters by using aeration & ozonation and two types of hollow-fiber membrane bioreactor (MBR) approaches. A secondary and more extensive objective derived from the first one is to draw a clearer, broader frame on the variation of physical-chemical parameters during the purification of wastewaters from food industry through different operating modes with the aim of improving the management of water purification process. Chemical oxygen demand (COD), pH, mixed liquor suspended solids (MLSS), total nitrogen, specific nitrogen (NH 4 + , NO 2 - , NO 3 - ) total phosphorous, and total surfactants were the measured parameters, and their influence was discussed in order to establish the best operating mode to achieve the purification performances. The integrated air-ozone aeration process applied in the second operating mode lead to a COD decrease by up to 90%, compared to only 75% obtained in a conventional biological activated sludge process. The combined purification process of MBR and ozonation produced an additional COD decrease of 10-15%, and made the Total Surfactants values to comply to the specific legislation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purification and some kinetic properties of catalase from parsley (Petroselinum hortense Hoffm., Apiaceae) leaves.

PubMed

Oztürk, Lokman; Bülbül, Metin; Elmastas, Mahfuz; Ciftçi, Mehmet

2007-01-01

In this study, catalase (CAT: EC 1.11.1.6) was purified from parsley (Petroselinum hortense) leaves; analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps, including preparation of homogenate, ammonium sulfate fractionation, and fractionation by DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 9.5% and had a specific activity of 1126 U (mg proteins)(-1). The overall purification was about 5.83-fold. A temperature of 4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured at 240 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acryl amide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for the enzyme. The molecular weight was found to be 183.29 kDa by Sephadex G-200 gel filtration chromatography. The stable pH, optimum pH, and ionic strength were determined for phosphate and Tris-HCl buffer systems. In addition, K(M) and V(max) values for H(2)O(2), at optimum pH and 25 degrees C, were determined by means of Lineweaver-Burk plots.

Halophilic viruses with varying biochemical and biophysical properties are amenable to purification with asymmetrical flow field-flow fractionation.

PubMed

Eskelin, Katri; Lampi, Mirka; Meier, Florian; Moldenhauer, Evelin; Bamford, Dennis H; Oksanen, Hanna M

2017-11-01

Viruses come in various shapes and sizes, and a number of viruses originate from extremities, e.g. high salinity or elevated temperature. One challenge for studying extreme viruses is to find efficient purification conditions where viruses maintain their infectivity. Asymmetrical flow field-flow fractionation (AF4) is a gentle native chromatography-like technique for size-based separation. It does not have solid stationary phase and the mobile phase composition is readily adjustable according to the sample needs. Due to the high separation power of specimens up to 50 µm, AF4 is suitable for virus purification. Here, we applied AF4 for extremophilic viruses representing four morphotypes: lemon-shaped, tailed and tailless icosahedral, as well as pleomorphic enveloped. AF4 was applied to input samples of different purity: crude supernatants of infected cultures, polyethylene glycol-precipitated viruses and viruses purified by ultracentrifugation. All four virus morphotypes were successfully purified by AF4. AF4 purification of culture supernatants or polyethylene glycol-precipitated viruses yielded high recoveries, and the purities were comparable to those obtained by the multistep ultracentrifugation purification methods. In addition, we also demonstrate that AF4 is a rapid monitoring tool for virus production in slowly growing host cells living in extreme conditions.

Simultaneous purification of DNA and RNA from microbiota in a single colonic mucosal biopsy.

PubMed

Moen, Aina E F; Tannæs, Tone M; Vatn, Simen; Ricanek, Petr; Vatn, Morten Harald; Jahnsen, Jørgen

2016-06-28

Nucleic acid purification methods are of importance when performing microbiota studies and especially when analysing the intestinal microbiota as we here find a wide range of different microbes. Various considerations must be taken to lyse the microbial cell wall of each microbe. In the present article, we compare several tissue lysis steps and commercial purification kits, to achieve a joint RNA and DNA purification protocol for the purpose of investigating the microbiota and the microbiota-host interactions in a single colonic mucosal tissue sample. A further optimised tissue homogenisation and lysis protocol comprising mechanical bead beating, lysis buffer replacement and enzymatic treatment, in combination with the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) resulted in efficient and simultaneous purification of microbial and human RNA and DNA from a single mucosal colonic tissue sample. The present work provides a unique possibility to study RNA and DNA from the same mucosal biopsy sample, making a direct comparison between metabolically active microbes and total microbial DNA. The protocol also offers an opportunity to investigate other members of a microbiota such as viruses, fungi and micro-eukaryotes, and moreover the possibility to extract data on microbiota and host interactions from one single mucosal biopsy.

Automated high-throughput purification of genomic DNA from plant leaf or seed using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2004-06-01

Three different methods of automated high throughput purification of genomic DNA from plant materials processed in 96 well plates are described. One method uses MagneSil paramagnetic particles to purify DNA present in single leaf punch samples or small seed samples, using 320ul capacity 96 well plates which minimizes reagent and plate costs. A second method uses 2.2 ml and 1.2 ml capacity plates and allows the purification of larger amounts of DNA from 5-6 punches of materials or larger amounts of seeds. The third method uses the MagneSil ONE purification system to purify a fixed amount of DNA, thus simplifying the processing of downstream applications by normalizing the amounts of DNA so they do not require quantitation. Protocols for the purification of a fixed yield of DNA, e.g. 1 ug, from plant leaf or seed samples using MagneSil paramagnetic particles and a Beckman-Coulter BioMek FX robot are described. DNA from all three methods is suitable for applications such as PCR, RAPD, STR, READIT SNP analysis, and multiplexed PCR systems. The MagneSil ONE system is also suitable for use with SNP detection systems such as Third Wave Technology"s Invader methods.

Application of hydrometallurgy techniques in quartz processing and purification: a review

NASA Astrophysics Data System (ADS)

Lin, Min; Lei, Shaomin; Pei, Zhenyu; Liu, Yuanyuan; Xia, Zhangjie; Xie, Feixiang

2018-04-01

Although there have been numerous studies on separation and purification of metallic minerals by hydrometallurgy techniques, applications of the chemical techniques in separation and purification of non-metallic minerals are rarely reported. This paper reviews disparate areas of study into processing and purification of quartz (typical non-metallic ore) in an attempt to summarize current work, as well as to suggest potential for future consolidation in the field. The review encompasses chemical techniques of the quartz processing including situations, progresses, leaching mechanism, scopes of application, advantages and drawbacks of micro-bioleaching, high temperature leaching, high temperature pressure leaching and catalyzed high temperature pressure leaching. Traditional leaching techniques including micro-bioleaching and high temperature leaching are unequal to demand of modern glass industry for quality of quartz concentrate because the quartz products has to be further processed. High temperature pressure leaching and catalyzed high temperature pressure leaching provide new ways to produce high-grade quartz sand with only one process and lower acid consumption. Furthermore, the catalyzed high temperature pressure leaching realizes effective purification of quartz with extremely low acid consumption (no using HF or any fluoride). It is proposed that, by integrating the different chemical processes of quartz processing and expounding leaching mechanisms and scopes of application, the research field as a monopolized industry would benefit.

Arginine homopeptides for plasmid DNA purification using monolithic supports.

PubMed

Cardoso, Sara; Sousa, Ângela; Queiroz, João A; Azzoni, Adriano R; Sousa, Fani

2018-06-15

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification. Copyright © 2018 Elsevier B.V. All rights reserved.

The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.

Retrovirus purification: method that conserves envelope glycoprotein and maximizes infectivity.

PubMed Central

McGrath, M; Witte, O; Pincus, T; Weissman, I L

1978-01-01

A Sepharose 4B chromatographic method for purification of retroviruses is described which was less time consuming, increased purified virus yields, conserved viral glycoprotein, and increased recovery of biological infectivity in comparison with conventional sucrose gradient ultracentrifugation techniques. Images PMID:205680

Beating Back Bacteria

NASA Technical Reports Server (NTRS)

1998-01-01

Under a NASA-Johnson Space Center contract, Umpqua Research developed the MCV (Trademark) (Microbial Check Valve) which uses iodinated ion exchange resin used for water purification systems aboard space missions. Using this resin, MRLB International, Inc., developed and commercialized the Dentapure purification cartridge used by dentists nationwide.

Purification Efficacy of Synthetic Cannabinoid Conjugates Using High-Pressure Liquid Chromatography

DTIC Science & Technology

In the current study, we successfully purified several synthetic cannabinoid (SC):dark quencher conjugates essential for the success of the synthetic... cannabinoid detection platform developed at the US Army Research Laboratorys Weapons and Materials Research Directorate. The purification was

Zein purification: the process, the product, market potential

USDA-ARS?s Scientific Manuscript database

The objectives of this article intend to give an overview of a zein purification, decolorization and deodorization process, methodologies to assess those properties and applications of the purified product. The process involves column filtration of commercial zein solutions through a combination of ...

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.

Phosphoric acid purification through different raw and activated clay materials (Southern Tunisia)

NASA Astrophysics Data System (ADS)

Trabelsi, Wafa; Tlili, Ali

2017-05-01

This study concerns the purification of Tunisian phosphoric acid produced by the Tunisian Chemical Group (TCG), using raw and activated clays materials from Southern Tunisia. The Gafsa basin clays samples (Jebel Hamadi (JHM); Jebel Stah (JS) and the El Hamma sample (Jebel Aïdoudi (JAD)) were activated with 3 M, HCl solution. Phosphoric acid purification was performed on raw and activated clays. Mineralogical characterisation was carried out using the X-ray powder diffraction method and infrared absorption spectroscopy. Textural changes between raw and activated clays were identified using SEM observations and specific surface analysis. Jebel Hamadi clays were almost dominated by smectite associated with kaolinite and illite traces, while Jebel Stah and Jebel Aïdoudi clays were composed of the association of smectite, illite and kaolinite. It is worth noting that the position of the smectite (001) reflection increased after the acidic activation in all studied samples, indicating the relaxation of the smectite structure along the c-axis. This was corroborated by the increasing specific surface area of the clay particles with the activation process. The specific surface area was close to 50 m2/g and 200 m2/g, for raw and activated materials, respectively. The maximum phosphoric acid purification was obtained by using activated clays with 3 N HCl for 4 h. This performance correlated with the maximum of the external specific surface area which generated strong acid sites. Furthermore, the best results of phosphoric acids purification from TCG were obtained at a specific consumption equivalent to 30 Kg of clay/ton of P2O5. These results showed that the best phosphoric acid purification was yielded by Jebel Aïdoudi clay. In all cases, the highest organic carbon reduction rates in the phosphoric acid after filtration were obtained at 90°C.

Pesticide removal from waste spray-tank water by organoclay adsorption after field application: an approach for a formulation of cyprodinil containing antifoaming/defoaming agents.

PubMed

Suciu, Nicoleta A; Ferrari, Tommaso; Ferrari, Federico; Trevisan, Marco; Capri, Ettore

2012-05-01

Many reports on purification of water containing pesticides are based on studies using unformulated active ingredients. However, most commercial formulations contain additives/adjuvants or are manufactured using microencapsulation which may influence the purification process. Therefore, the main objective of this work was to develop and test a pilot scheme for decontaminating water containing pesticides formulated with antifoaming/defoaming agents. The Freundlich adsorption coefficients of formulation of cyprodinil, a new-generation fungicide, onto the organoclay Cloisite 20A have been determined in the laboratory in order to predict the efficiency of this organoclay in removing the fungicide from waste spray-tank water. Subsequently, the adsorption tests were repeated in the pilot system in order to test the practical operation of the purification scheme. The laboratory adsorption tests successfully predicted the efficiency of the pilot purification system, which removed more than 96% cyprodinil over a few hours. The passing of the organoclay-cyprodinil suspension through a layer of biomass gave 100% recovery of the organoclay at the surface of the biomass after 1 week. The organoclay was composted after the treatment to try to break down the fungicide so as to allow safe disposal of the waste, but cyprodinil was not significantly dissipated after 90 days. The purification scheme proved to be efficient for decontaminating water containing cyprodinil formulated with antifoaming/defoaming agents, but additional treatments for the adsorbed residues still appear to be necessary even for a moderately persistent pesticide such as cyprodinil. Furthermore, a significant conclusion of this study concerns the high influence of pesticide formulations on the process of purification of water containing these compounds, which should be taken into account when developing innovative decontamination schemes, especially for practical applications.

Immobilized Metal Affinity Chromatography Co-Purifies TGF-β1 with Histidine-Tagged Recombinant Extracellular Proteins

PubMed Central

Kaur, Jasvir; Reinhardt, Dieter P.

2012-01-01

Extracellular recombinant proteins are commonly produced using HEK293 cells as histidine-tagged proteins facilitating purification by immobilized metal affinity chromatography (IMAC). Based on gel analyses, this one-step purification typically produces proteins of high purity. Here, we analyzed the presence of TGF-β1 in such IMAC purifications using recombinant extracellular fibrillin-1 fragments as examples. Analysis of various purified recombinant fibrillin-1 fragments by ELISA consistently revealed the presence of picomolar concentrations of active and latent TGF-β1, but not of BMP-2. These quantities of TGF-β1 were not detectable by Western blotting and mass spectrometry. However, the amounts of TGF-β1 were sufficient to consistently trigger Smad2 phosphorylation in fibroblasts. The purification mechanism was analyzed to determine whether the presence of TGF-β1 in these protein preparations represents a specific or non-specific co-purification of TGF-β1 with fibrillin-1 fragments. Control purifications using conditioned medium from non-transfected 293 cells yielded similar amounts of TGF-β1 after IMAC. IMAC of purified TGF-β1 and the latency associated peptide showed that these proteins bound to the immobilized nickel ions. These data clearly demonstrate that TGF-β1 was co-purified by specific interactions with nickel, and not by specific interactions with fibrillin-1 fragments. Among various chromatographic methods tested for their ability to eliminate TGF-β1 from fibrillin-1 preparations, gel filtration under high salt conditions was highly effective. As various recombinant extracellular proteins purified in this fashion are frequently used for experiments that can be influenced by the presence of TGF-β1, these findings have far-reaching implications for the required chromatographic schemes and quality controls. PMID:23119075

TlBr purification and single crystal growth for the detector applications

NASA Astrophysics Data System (ADS)

Kozlov, Vasilij; Heikkilä, Mikko; Kostamo, Pasi; Lipsanen, Harri; Leskelä, Markku

2011-05-01

The combination of distillation, Bridgman-Stockbarger, hydrothermal recrystallisation and travelling molten zone (TMZ) methods were used for TlBr purification. Grown crystals were characterised by XRD rocking curve and FTIR spectroscopy methods, and by electrical measurements made from 200 to 300 K.

A general method for the purification of restriction enzymes.

PubMed Central

Greene, P J; Heyneker, H L; Bolivar, F; Rodriguez, R L; Betlach, M C; Covarrubias, A A; Backman, K; Russel, D J; Tait, R; Boyer, H W

1978-01-01

An abbreviated procedure has been developed for the purification of restriction endonucleases. This procedure uses chromatography on phosphocellulose and hydroxylapatite and results in enzymes of sufficient purity to permit their use in the sequencing, molecular cloning, and physical mapping of DNA. PMID:673857

Efficient stable isotope labeling and purification of vitamin D receptor from inclusion bodies

PubMed Central

Zhu, Jinge; Rao, Hongyu; Tonelli, Marco; Westler, Milo; Singarapu, Kiran K.; Markley, John L.; DeLuca, Hector F.; Assadi-Porter, Fariba M.

2012-01-01

Vitamin D receptor (VDR) plays a crucial role in many cellular processes including calcium and phosphate homeostasis. Previous purification methods from prokaryotic and eukaryotic expression systems were challenged by low protein solubility accompanied by multi purification steps resulting in poor protein recovery. The full-length VDR and its ligand binding domain (LBD) were mostly (>90%) insoluble even when expressed at low temperatures in the bacterial system. We describe a one-step procedure that results in the purification of rat VDR and LBD proteins in high-yield from E. coli inclusion bodies. The heterologously expressed protein constructs retain full function as demonstrated by ligand binding and DNA binding assays. Furthermore, we describe an efficient strategy for labeling these proteins with, 13C, and 15N for structural and functional studies by nuclear magnetic resonance (NMR) spectroscopy. This efficient production system will facilitate future studies on the mechanism of vitamin D action including characterization of the large number of synthetic vitamin D analogs that have been developed. PMID:22750673

Application of alkaline solid residue of electric arc furnace dust for neutralization/purification of electroplating wastewaters.

PubMed

Elez, Loris; Orescanin, Visnja; Sofilic, Tahir; Mikulic, Nenad; Ruk, Damir

2008-10-01

The purpose of this work was development of an appropriate procedure for the neutralization/purification of electroplating wastewater (EWW) with alkaline solid residue (ASR) by-product of the alkaline extraction of zinc and lead from electric arc furnace dust (EAFD). Removal efficiency of ASR at optimum purification conditions (pH 8 and mixing time; 20 minutes) for the elements Pb, Cr (VI), Cr (III), Fe, Ni, Cu and Zn were 94.92%, 97.58%, 99.59%, 99.48%, 97.25% and 99.97%, respectively. The concentrations of all elements in the purified wastewater were significantly lower in relation to the upper permissible limit for wastewaters suitable for discharge into the environment. The remaining waste mud was regenerated in the strong alkaline medium and successfully applied once again for the neutralization/purification of EWW. Removal efficiencies of heavy metals accomplished with regenerated waste mud were comparable to these achieved by original ASR. Elemental concentrations in the leachates of the waste mud were in accordance with regulated values.

Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330

Tandem SUMO fusion vectors for improving soluble protein expression and purification.

PubMed

Guerrero, Fernando; Ciragan, Annika; Iwaï, Hideo

2015-12-01

Availability of highly purified proteins in quantity is crucial for detailed biochemical and structural investigations. Fusion tags are versatile tools to facilitate efficient protein purification and to improve soluble overexpression of proteins. Various purification and fusion tags have been widely used for overexpression in Escherichia coli. However, these tags might interfere with biological functions and/or structural investigations of the protein of interest. Therefore, an additional purification step to remove fusion tags by proteolytic digestion might be required. Here, we describe a set of new vectors in which yeast SUMO (SMT3) was used as the highly specific recognition sequence of ubiquitin-like protease 1, together with other commonly used solubility enhancing proteins, such as glutathione S-transferase, maltose binding protein, thioredoxin and trigger factor for optimizing soluble expression of protein of interest. This tandem SUMO (T-SUMO) fusion system was tested for soluble expression of the C-terminal domain of TonB from different organisms and for the antiviral protein scytovirin. Copyright © 2015 Elsevier Inc. All rights reserved.

Plasmid DNA production combining antibiotic-free selection, inducible high yield fermentation, and novel autolytic purification.

PubMed

Carnes, Aaron E; Hodgson, Clague P; Luke, Jeremy M; Vincent, Justin M; Williams, James A

2009-10-15

DNA vaccines and gene medicines, derived from bacterial plasmids, are emerging as an important new class of pharmaceuticals. However, the challenges of performing cell lysis processes for plasmid DNA purification at an industrial scale are well known. To address downstream purification challenges, we have developed autolytic Escherichia coli host strains that express endolysin (phage lambdaR) in the cytoplasm. Expression of the endolysin is induced during fermentation by a heat inducible promoter. The endolysin remains in the cytoplasm, where it is separated from its peptidoglycan substrate in the cell wall; hence the cells remain alive and intact and can be harvested by the usual methods. The plasmid DNA is then recovered by autolytic extraction under slightly acidic, low salt buffer conditions and treatment with a low concentration of non-ionic detergent. Under these conditions the E. coli genomic DNA remains associated with the insoluble cell debris and is removed by a solid-liquid separation. Here, we report fermentation, lysis methods, and plasmid purification using autolytic hosts.

SwellGel: an affinity chromatography technology for high-capacity and high-throughput purification of recombinant-tagged proteins.

PubMed

Draveling, C; Ren, L; Haney, P; Zeisse, D; Qoronfleh, M W

2001-07-01

The revolution in genomics and proteomics is having a profound impact on drug discovery. Today's protein scientist demands a faster, easier, more reliable way to purify proteins. A high capacity, high-throughput new technology has been developed in Perbio Sciences for affinity protein purification. This technology utilizes selected chromatography media that are dehydrated to form uniform aggregates. The SwellGel aggregates will instantly rehydrate upon addition of the protein sample, allowing purification and direct performance of multiple assays in a variety of formats. SwellGel technology has greater stability and is easier to handle than standard wet chromatography resins. The microplate format of this technology provides high-capacity, high-throughput features, recovering milligram quantities of protein suitable for high-throughput screening or biophysical/structural studies. Data will be presented applying SwellGel technology to recombinant 6x His-tagged protein and glutathione-S-transferase (GST) fusion protein purification. Copyright 2001 Academic Press.

High-yield recombinant expression and purification of marginally soluble, short elastin-like polypeptides.

PubMed

Bahniuk, Markian S; Alshememry, Abdullah K; Unsworth, Larry D

2016-12-01

The protocol described here is designed as an extension of existing techniques for creating elastin-like polypeptides. It allows for the expression and purification of elastin-like polypeptide (ELP) constructs that are poorly expressed or have very low transition temperatures. DNA concatemerization has been modified to reduce issues caused by methylation sensitivity and inefficient cloning. Linearization of the modified expression vector has been altered to greatly increase cleavage efficiency. The purification regimen is based upon using denaturing metal affinity chromatography to fully solubilize and, if necessary, pre-concentrate the target peptide before purification by inverse temperature cycling (ITC). This protocol has been used to express multiple leucine-containing elastin-like polypeptides, with final yields of 250-660 mg per liter of cells, depending on the specific construct. This was considerably greater than previously reported yields for similar ELPs. Due to the relative hydrophobicity of the tested constructs, even compared with commonly employed ELPs, conventional methods would not have been able to be purify these peptides.

Purification-Free, Target-Selective Immobilization of a Protein from Cell Lysates.

PubMed

Cha, Jaehyun; Kwon, Inchan

2018-02-27

Protein immobilization has been widely used for laboratory experiments and industrial processes. Preparation of a recombinant protein for immobilization usually requires laborious and expensive purification steps. Here, a novel purification-free, target-selective immobilization technique of a protein from cell lysates is reported. Purification steps are skipped by immobilizing a target protein containing a clickable non-natural amino acid (p-azidophenylalanine) in cell lysates onto alkyne-functionalized solid supports via bioorthogonal azide-alkyne cycloaddition. In order to achieve a target protein-selective immobilization, p-azidophenylalanine was introduced into an exogenous target protein, but not into endogenous non-target proteins using host cells with amber codon-free genomic DNAs. Immobilization of superfolder fluorescent protein (sfGFP) from cell lysates is as efficient as that of the purified sfGFP. Using two fluorescent proteins (sfGFP and mCherry), the authors also demonstrated that the target proteins are immobilized with a minimal immobilization of non-target proteins (target-selective immobilization). © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Application of QUAL2K Model to Assess Ecological Purification Technology for a Polluted River

PubMed Central

Zhu, Wenting; Niu, Qian; Zhang, Ruibin; Ye, Rui; Qian, Xin; Qian, Yu

2015-01-01

Industrialization and urbanization have caused water pollution and ecosystem degradation, especially in urban canals and rivers in China; accordingly, effective water quality improvement programs are needed. In this study, the Tianlai River in Jiangsu, China was taken as a research site, and a combination of ecological purification technologies consisting of biological rope, phytoremediation, and activated carbon were applied in a laboratory-scale study to examine degradation coefficients under dynamic water conditions. Coefficients were then input into the QUAL2K model to simulate various hypothetical scenarios and determine the minimum density of ecological purification combination and hydraulic retention time (HRT) to meet Grade V or IV of the China standard for surface water. The minimum densities for Grade V and IV were 1.6 times and 2 times the experimental density, while the minimum HRTs for Grade V and IV were 2.4 day and 3 day. The results of this study should provide a practical and efficient design method for ecological purification programs. PMID:25689997

Synergistic effect of Brønsted acid and platinum on purification of automobile exhaust gases

PubMed Central

Fu, Wei; Li, Xin-Hao; Bao, Hong-Liang; Wang, Kai-Xue; Wei, Xiao; Cai, Yi-Yu; Chen, Jie-Sheng

2013-01-01

The catalytic purification of automobile exhaust gases (CO, NOx and hydrocarbons) is one of the most practiced conversion processes used to lower the emissions and to reduce the air pollution. Nevertheless, the good performance of exhaust gas purification catalysts often requires the high consumption of noble metals such as platinum. Here we report that the Brønsted acid sites on the external surface of a microporous silicoaluminophosphate (SAPO) act as a promoter for exhaust gas purification, effectively cutting the loading amount of platinum in the catalyst without sacrifice of performance. It is revealed that in the Pt-loaded SAPO-CHA catalyst, there exists a remarkable synergistic effect between the Brønsted acid sites and the Pt nanoparticles, the former helping to adsorb and activate the hydrocarbon molecules for NO reduction during the catalytic process. The thermal stability of SAPO-CHA also makes the composite catalyst stable and reusable without activity decay. PMID:23907148

Peat Water Purification by Hydroxyapatite (HAp) Synthesized from Waste Pensi (Corbicula moltkiana) Shells

NASA Astrophysics Data System (ADS)

Fajri Alif, Matlal; Aprillia, Wandha; Arief, Syukri

2018-01-01

Hydroxyapatite (HAP) were synthesized from Pensi (Corbicula moltkiana) sheels by hydrothermal method and used as adsorbent for peat water purification. Batch adsorption experiments were performed to investigate the effects of various factors such as contact time, adsorbent dosage, and pH. The obtained materials were characterized by powder X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and scanning electron microscope (SEM). Results showed that HAP calcined at 900°C (HAP900) and 1000°C (HAP1000) have a poorly crystalline shape. HAP900 also contain Tetracalsium Phosphate (TTCP) with a Ca/P molar ratio 2.18, while HAP 1000 contain HAp with a Ca/P molar ratio 1.67. Optimum condition for peat water purification with HAP900 and HAP1000 were both achieved at 1 hours, 1 grams adsorben mass at pH 2. SEM micrographs show that after purification, the surface of HAP were covered by organic compounds from peat water.

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

Synergistic effect of Brønsted acid and platinum on purification of automobile exhaust gases.

PubMed

Fu, Wei; Li, Xin-Hao; Bao, Hong-Liang; Wang, Kai-Xue; Wei, Xiao; Cai, Yi-Yu; Chen, Jie-Sheng

2013-01-01

The catalytic purification of automobile exhaust gases (CO, NOx and hydrocarbons) is one of the most practiced conversion processes used to lower the emissions and to reduce the air pollution. Nevertheless, the good performance of exhaust gas purification catalysts often requires the high consumption of noble metals such as platinum. Here we report that the Brønsted acid sites on the external surface of a microporous silicoaluminophosphate (SAPO) act as a promoter for exhaust gas purification, effectively cutting the loading amount of platinum in the catalyst without sacrifice of performance. It is revealed that in the Pt-loaded SAPO-CHA catalyst, there exists a remarkable synergistic effect between the Brønsted acid sites and the Pt nanoparticles, the former helping to adsorb and activate the hydrocarbon molecules for NO reduction during the catalytic process. The thermal stability of SAPO-CHA also makes the composite catalyst stable and reusable without activity decay.

Integrated Method for Purification and Single-Particle Characterization of Lentiviral Vector Systems by Size Exclusion Chromatography and Tunable Resistive Pulse Sensing.

PubMed

Heider, Susanne; Muzard, Julien; Zaruba, Marianne; Metzner, Christoph

2017-07-01

Elements derived from lentiviral particles such as viral vectors or virus-like particles are commonly used for biotechnological and biomedical applications, for example in mammalian protein expression, gene delivery or therapy, and vaccine development. Preparations of high purity are necessary in most cases, especially for clinical applications. For purification, a wide range of methods are available, from density gradient centrifugation to affinity chromatography. In this study we have employed size exclusion columns specifically designed for the easy purification of extracellular vesicles including exosomes. In addition to viral marker protein and total protein analysis, a well-established single-particle characterization technology, termed tunable resistive pulse sensing, was employed to analyze fractions of highest particle load and purity and characterize the preparations by size and surface charge/electrophoretic mobility. With this study, we propose an integrated platform combining size exclusion chromatography and tunable resistive pulse sensing for monitoring production and purification of viral particles.

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.

Quantification and characterisation of fatty acid methyl esters in microalgae: Comparison of pretreatment and purification methods.

PubMed

Lage, Sandra; Gentili, Francesco G

2018-06-01

A systematic qualitative and quantitative analysis of fatty acid methyl esters (FAMEs) is crucial for microalgae species selection for biodiesel production. The aim of this study is to identify the best method to assess microalgae FAMEs composition and content. A single-step method, was tested with and without purification steps-that is, separation of lipid classes by thin-layer chromatography (TLC) or solid-phase extraction (SPE). The efficiency of a direct transesterification method was also evaluated. Additionally, the yield of the FAMEs and the profiles of the microalgae samples with different pretreatments (boiled in isopropanol, freezing, oven-dried and freeze-dried) were compared. The application of a purification step after lipid extraction proved to be essential for an accurate FAMEs characterisation. The purification methods, which included TLC and SPE, provided superior results compared to not purifying the samples. Freeze-dried microalgae produced the lowest FAMEs yield. However, FAMEs profiles were generally equivalent among the pretreatments. Copyright © 2018 Elsevier Ltd. All rights reserved.

Purification and characterization of broccoli (Brassica oleracea var. italica) myrosinase (β-thioglucosidase glucohydrolase).

PubMed

Mahn, Andrea; Angulo, Alejandro; Cabañas, Fernanda

2014-12-03

Myrosinase (β-thioglucosidase glucohydrolase, EC 3.2.1.147) from broccoli (Brassica oleracea var. italica) was purified by ammonium sulfate precipitation followed by concanavalin A affinity chromatography, with an intermediate dialysis step, resulting in 88% recovery and 1318-fold purification. These are the highest values reported for the purification of any myrosinase. The subunits of broccoli myrosinase have a molecular mass of 50-55 kDa. The native molecular mass of myrosinase was 157 kDa, and accordingly, it is composed of three subunits. The maximum activity was observed at 40 °C and at pH below 5.0. Kinetic assays demonstrated that broccoli myrosinase is subjected to substrate (sinigrin) inhibition. The Michaelis-Menten model, considering substrate inhibition, gave Vmax equal to 0.246 μmol min(-1), Km equal to 0.086 mM, and K(I) equal to 0.368 mM. This is the first study about purification and characterization of broccoli myrosinase.

[Isolation and purification of alpha-glycerophosphate oxidase in a polyethylene glycol/(NH4 )2SO4 aqueous two-phase system].

PubMed

Meng, Yao; Jin, Jiagui; Liu, Shuangfeng; Yang, Min; Zhang, Qinglian; Wan, Li; Tang, Kun

2014-02-01

Alpha-glycerophosphate oxidase (alpha-GPO) from Enterococcus casseli flavus was successfully isolated and purified by using polyethylene glycol (PEG)/(NH4)2SO4 aqueous two-phase system (ATPS). The results showed that the chosen PEG/(NH4)2SO4 ATPS could be affected by PEG molecular weight, pH, concentration of PEG and (NH4)2SO4, and inorganic salt as well as additional amount of crude enzyme. After evaluating these influencing factors, the final optimum purification strategy was formed by 16.5% (m/m) PEG2000, 13.2% (m/m) (NH4)2SO4, pH 7.5 and 30% (m/m) additive crude enzyme, respectively. The NaCl was a negative influencing factor which would lead to lower purification fold and activity recovery. These conditions eventually resulted in the activity recovery of 89% (m/m), distribution coefficient of 1.2 and purification fold of 7.0.

Overview of the purification of recombinant proteins.

PubMed

Wingfield, Paul T

2015-04-01

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 throughout Current Protocols in Protein Science. In the interim, there has been a shift toward 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, other commonly used expression systems are mentioned and, apart from cell-breakage methods, protein purification methods and strategies are essentially the same. Copyright © 2015 John Wiley & Sons, Inc.

Separation and purification of enzymes by continuous pH-parametric pumping

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, S.Y.; Lin, C.K.; Juang, L.Y.

1985-10-01

Trypsin and chymotrypsin were separated from porcine pancreas extract by continuous pH-parametric pumping. CHOM (chicken ovomucoid) was convalently bound to laboratory-prepared crab chitin with glutaraldehyde to form an affinity adsorbent of trypsin. The pH levels of top and bottom feeds were 8.0 and 2.5, respectively. Similar inhibitor, DKOM (duck ovomucoid), and pH levels 8.0 and 2.0 for top and bottom feeds, respectively, were used for separation and purification of chymotrypsin. e-Amino caproyl-D-tryptophan methyl ester was coupled to chitosan to form an affinity adsorbent for stem bromelain. The pH levels were 8.7 and 3.0. Separation continued fairly well with high yield,more » e.g., 95% recovery of trypsin after continuous pumping of 10 cycles. Optimum operational conditions for concentration and purification of these enzymes were investigated. The results showed that the continuous pH-parametric pumping coupled with affinity chromatography is effective for concentration and purification of enzymes. 19 references.« less

Optimization of a micro-scale, high throughput process development tool and the demonstration of comparable process performance and product quality with biopharmaceutical manufacturing processes.

PubMed

Evans, Steven T; Stewart, Kevin D; Afdahl, Chris; Patel, Rohan; Newell, Kelcy J

2017-07-14

In this paper, we discuss the optimization and implementation of a high throughput process development (HTPD) tool that utilizes commercially available micro-liter sized column technology for the purification of multiple clinically significant monoclonal antibodies. Chromatographic profiles generated using this optimized tool are shown to overlay with comparable profiles from the conventional bench-scale and clinical manufacturing scale. Further, all product quality attributes measured are comparable across scales for the mAb purifications. In addition to supporting chromatography process development efforts (e.g., optimization screening), comparable product quality results at all scales makes this tool is an appropriate scale model to enable purification and product quality comparisons of HTPD bioreactors conditions. The ability to perform up to 8 chromatography purifications in parallel with reduced material requirements per run creates opportunities for gathering more process knowledge in less time. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Topic model-based mass spectrometric data analysis in cancer biomarker discovery studies.

PubMed

Wang, Minkun; Tsai, Tsung-Heng; Di Poto, Cristina; Ferrarini, Alessia; Yu, Guoqiang; Ressom, Habtom W

2016-08-18

A fundamental challenge in quantitation of biomolecules for cancer biomarker discovery is owing to the heterogeneous nature of human biospecimens. Although this issue has been a subject of discussion in cancer genomic studies, it has not yet been rigorously investigated in mass spectrometry based proteomic and metabolomic studies. Purification of mass spectometric data is highly desired prior to subsequent analysis, e.g., quantitative comparison of the abundance of biomolecules in biological samples. We investigated topic models to computationally analyze mass spectrometric data considering both integrated peak intensities and scan-level features, i.e., extracted ion chromatograms (EICs). Probabilistic generative models enable flexible representation in data structure and infer sample-specific pure resources. Scan-level modeling helps alleviate information loss during data preprocessing. We evaluated the capability of the proposed models in capturing mixture proportions of contaminants and cancer profiles on LC-MS based serum proteomic and GC-MS based tissue metabolomic datasets acquired from patients with hepatocellular carcinoma (HCC) and liver cirrhosis as well as synthetic data we generated based on the serum proteomic data. The results we obtained by analysis of the synthetic data demonstrated that both intensity-level and scan-level purification models can accurately infer the mixture proportions and the underlying true cancerous sources with small average error ratios (<7 %) between estimation and ground truth. By applying the topic model-based purification to mass spectrometric data, we found more proteins and metabolites with significant changes between HCC cases and cirrhotic controls. Candidate biomarkers selected after purification yielded biologically meaningful pathway analysis results and improved disease discrimination power in terms of the area under ROC curve compared to the results found prior to purification. We investigated topic model-based inference methods to computationally address the heterogeneity issue in samples analyzed by LC/GC-MS. We observed that incorporation of scan-level features have the potential to lead to more accurate purification results by alleviating the loss in information as a result of integrating peaks. We believe cancer biomarker discovery studies that use mass spectrometric analysis of human biospecimens can greatly benefit from topic model-based purification of the data prior to statistical and pathway analyses.

Purification of Plant Receptor Kinases from Plant Plasma Membranes.

PubMed

Lee, Jin Suk

2017-01-01

Receptor kinases play a central role in various biological processes, but due to their low abundance and highly hydrophobic and dynamic nature, only a few of them have been functionally characterized, and their partners and ligands remain unidentified. Receptor protein extraction and purification from plant tissues is one of the most challenging steps for the success of various biochemical analyses to characterize their function. Immunoprecipitation is a widely used and selective method for enriching or purifying a specific protein. Here we describe two different optimized protein purification protocols, batch and on-chip immunoprecipitation, which efficiently isolate plant membrane receptor kinases for functional analysis.

Purification of oily wastewater by hybrid UF/MD.

PubMed

Gryta, M; Karakulski, K; Morawski, A W

2001-10-01

Investigations on the treatment of oily wastewater by a combination of ultrafiltration (UF) and membrane distillation (MD) as a final purification method have been performed. A tubular UF module equipped with polyvinylidene fluoride (PVDF) membranes and a capillary MD module with polypropylene membranes were tested using a typical bilge water collected from a harbour without pretreatment. The permeate obtained from the UF process generally contains less than 5 ppm of oil. A further purification of the UF permeate by membrane distillation results in a complete removal of oil from wastewater and a very high reduction of the total organic carbon (99.5%) and total dissolved solids (99.9%).

[Optimizing the extracting technique of ampelopsin from Ampelopsis cantoniensis Planch by a uniform design method].

PubMed

He, Zhi-feng; Zeng, Sa; Hou, Juan-juan; Liu, De-yu

2006-07-01

To optimize the preparation of ampelopsin from Ampelopsis Cantoniensis Planch. The extraction and purification process was studied by the uniform design with the extract of ampelopsin content and purity as markers. The facters which influence the extraction and the purification of ampelopsin content were studied by uniform design. The optimum extraction and purification process: the concentration for alcohol was 90%, and refluxing quartic, 1.5 h each time; extraction by petroleum ether quintic, the mount of active carbon was 1 g/100 g of the medicine material, and recrystaling thrice. This extraction process has higher yield of ampelopsin and is available for production.

A gradient-free method for the purification of infective dengue virus for protein-level investigations.

PubMed

Jensen, Stephanie M; Nguyen, Celina T; Jewett, John C

2016-09-01

Dengue virus (DENV) is a mosquito-transmitted flavivirus that infects approximately 100 million people annually. Multi-day protocols for purification of DENV reduce the infective titer due to viral sensitivity to both temperature and pH. Herein we describe a 5-h protocol for the purification of all DENV serotypes, utilizing traditional gradient-free ultracentrifugation followed by selective virion precipitation. This protocol allows for the separation of DENV from contaminating proteins - including intact C6/36 densovirus, for the production of infective virus at high concentration for protein-level analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Separation and purification of thymopentin with molecular imprinting membrane by solid phase extraction disks.

PubMed

Wang, Chaoli; Hu, Xiaoling; Guan, Ping; Wu, Danfeng; Qian, Liwei; Li, Ji; Song, Renyuan

2015-01-01

The synthesis and performance of molecularly imprinted membranes (MIMs) as a solid phase extraction packing materials for the separation and purification of thymopentin from crude samples was described. In order to increase structural selectivity and imprinting efficiency, surface-initiated ATRP and ionic liquid (1-vinyl-3-ethyl acetate imidazolium chloride) were used to prepare molecularly imprinting membranes. The results demonstrated that solid phase extraction disks stuffed by MIMs with ionic liquids as functional monomer demonstrated high isolation and purification of performance to the thymopentin. The molecular recognition of thymopentin was analyzed by using molecular modeling software. Copyright © 2014 Elsevier B.V. All rights reserved.

Purification of Logic-Qubit Entanglement.

PubMed

Zhou, Lan; Sheng, Yu-Bo

2016-07-05

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.

Electrophoretic cell separation using microspheres. [purification of lymphocytes

NASA Technical Reports Server (NTRS)

Smolka, A.; Sachs, G.

1980-01-01

Methods of cell separation based on the electrokinetic properties of the cell membrane offer a degree of discrimination among cell populations which is not available with methods based on cell size or density alone. Studies aimed at extending red cell separations using microspheres to purification of lymphocytes.

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

USDA-ARS?s Scientific Manuscript database

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...

Purification of Rubisco Activase from Leaves or after Expression in Escherichia coli.

USDA-ARS?s Scientific Manuscript database

Rubisco activase is a molecular chaperone that modulates the activation state of Rubisco by catalyzing the ATP-dependent removal of tightly-bound inhibitory sugar-phosphates from Rubisco’s catalytic sites. This chapter reports methods developed for the purification of native and recombinant Rubisco...

Extraction, Purification, and Spectroscopic Characterization of a Mixture of Capsaicinoids

ERIC Educational Resources Information Center

Wagner, Carl E.; Cahill, Thomas M.; Marshall, Pamela A.

2011-01-01

This laboratory experiment provides a safe and effective way to instruct undergraduate organic chemistry students about natural-product extraction, purification, and NMR spectroscopic characterization. On the first day, students extract dried habanero peppers with toluene, perform a pipet silica gel column to separate carotenoids from…

ELISA AND SOL-GEL BASED IMMUNOAFFINITY PURIFICATION OF THE PYRETHROID BIOALLETHRIN IN FOOD AND ENVIRONMENTAL SAMPLES

EPA Science Inventory

The peer-reviewed article describes the development of a new sol-gel based immunoaffinity purification procedure and an immunoassay for the pyrethroid bioallethrin. The immunoaffinity chromatography procedure was applied to food samples providing an efficient cleanup prior to im...

40 CFR 61.62 - Emission standard for ethylene dichloride plants.

Code of Federal Regulations, 2013 CFR

2013-07-01

...) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS National Emission... dichloride purification. The concentration of vinyl chloride in each exhaust gas stream from any equipment used in ethylene dichloride purification is not to exceed 10 ppm (average for 3-hour period), except as...

40 CFR 61.62 - Emission standard for ethylene dichloride plants.

Code of Federal Regulations, 2014 CFR

2014-07-01

...) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS National Emission... dichloride purification. The concentration of vinyl chloride in each exhaust gas stream from any equipment used in ethylene dichloride purification is not to exceed 10 ppm (average for 3-hour period), except as...

40 CFR 61.62 - Emission standard for ethylene dichloride plants.

Code of Federal Regulations, 2012 CFR

2012-07-01

...) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS National Emission... dichloride purification. The concentration of vinyl chloride in each exhaust gas stream from any equipment used in ethylene dichloride purification is not to exceed 10 ppm (average for 3-hour period), except as...

40 CFR 61.62 - Emission standard for ethylene dichloride plants.

Code of Federal Regulations, 2011 CFR

2011-07-01

...) AIR PROGRAMS (CONTINUED) NATIONAL EMISSION STANDARDS FOR HAZARDOUS AIR POLLUTANTS National Emission... dichloride purification. The concentration of vinyl chloride in each exhaust gas stream from any equipment used in ethylene dichloride purification is not to exceed 10 ppm (average for 3-hour period), except as...

Nanofluidic Lab-On-Chip Technology for DNA Identification

DTIC Science & Technology

2013-09-30

samples Fluorescently labeled (FAM tag) DNA oligomers (10, 20, and 50 bases long) were purchased with standard desalting and additional HPLC purification...A.2 DNA samples: DNA oligomers (10, 20, 50 nt long) were purchased with standard desalting and additional HPLC purification for the 50 base

Purification of crime scene DNA extracts using centrifugal filter devices

PubMed Central

2013-01-01

Background The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. Methods Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. Results Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. Conclusions Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used. PMID:23618387

High-throughput purification of recombinant proteins using self-cleaving intein tags.

PubMed

Coolbaugh, M J; Shakalli Tang, M J; Wood, D W

2017-01-01

High throughput methods for recombinant protein production using E. coli typically involve the use of affinity tags for simple purification of the protein of interest. One drawback of these techniques is the occasional need for tag removal before study, which can be hard to predict. In this work, we demonstrate two high throughput purification methods for untagged protein targets based on simple and cost-effective self-cleaving intein tags. Two model proteins, E. coli beta-galactosidase (βGal) and superfolder green fluorescent protein (sfGFP), were purified using self-cleaving versions of the conventional chitin-binding domain (CBD) affinity tag and the nonchromatographic elastin-like-polypeptide (ELP) precipitation tag in a 96-well filter plate format. Initial tests with shake flask cultures confirmed that the intein purification scheme could be scaled down, with >90% pure product generated in a single step using both methods. The scheme was then validated in a high throughput expression platform using 24-well plate cultures followed by purification in 96-well plates. For both tags and with both target proteins, the purified product was consistently obtained in a single-step, with low well-to-well and plate-to-plate variability. This simple method thus allows the reproducible production of highly pure untagged recombinant proteins in a convenient microtiter plate format. Copyright © 2016 Elsevier Inc. All rights reserved.

Specific solubilization of impurities in culture media: Arg solution improves purification of pH-responsive tag CspB50 with Teriparatide.

PubMed

Oki, Shogo; Nonaka, Takahiro; Shiraki, Kentaro

2018-06-01

Protein purification using non-chromatographic methods is a simple technique that avoids costly resin. Recently, a cell surface protein B (CspB) tag has been developed for a pH-responsive tag for protein purification by solid-liquid separation. Proteins fused with the CspB tag show reversible insolubilization at acidic pH that can be used in solid-liquid separation for protein purification. However, brown-color impurities from co-precipitation hamper further analysis of the target proteins. In this study, we investigated the effect of additives on the co-precipitation of CspB-tagged Teriparatide (CspB50TEV-Teriparatide) expressed in Corynebacterium glutamicum and associated impurities. Arginine (Arg) at 1.0 M was found to be the most effective additive for removing impurities, particularly carotenoids and nucleic acids. Furthermore, all impurities detected in the fluorescence and absorbance spectra were successfully removed by the repetition of precipitation-redissolution in the Arg solution. The precipitation yield of the CspB50TEV-Teriparatide did not change with the addition of Arg and the repetition of the precipitation-redissolution process. Collectively, our findings indicate that the specific desorption of π-electron rich compounds by Arg may be useful in conjunction with the pH-responsive CspB tag for solid-liquid protein purification. Copyright © 2018 Elsevier Inc. All rights reserved.

Process and economic evaluation of the extraction and purification of recombinant beta-glucuronidase from transgenic corn

PubMed

Evangelista; Kusnadi; Howard; Nikolov

1998-07-01

A process model for the recovery and purification of recombinant beta-glucuronidase (rGUS) from transgenic corn was developed, and the process economics were estimated. The base-case bioprocessing plant operates 7500 h/year processing 1.74 million (MM) kg of transgenic corn containing 0.015% (db) rGUS. The process consists of milling the corn into flour, extraction of protein by using 50 mM sodium phosphate buffer, and rGUS purification by ion exchange and hydrophobic interaction chromatography. About 137 kg of rGUS of 83% (db) purity can be produced annually. The production cost amounted to $43 000/kg of rGUS. The cost of milling, protein extraction, and rGUS purification accounted for 6, 40, and 48% of annual operating cost, respectively. The cost of transgenic corn was 31% of the raw material costs or 6% of the annual operating cost. About 78% of the cost of buffer and water were incurred in the protein extraction section, while 88% of other consumables were from the purification section. The sensitivity analysis indicated that rGUS can be produced profitably from corn even at the 0.015% (db) expression level, assuming a selling price of $100 000/kg GUS. An increase in rGUS expression levels up to 0.08% significantly improves the process economics.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

PubMed

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

Affinity Purification of Tumor Necrosis Factor-α Expressed in Raji Cells by Produced scFv Antibody Coupled CNBr-Activated Sepharose

PubMed Central

Abdolalizadeh, Jalal; Majidi Zolbanin, Jafar; Nouri, Mohammad; Baradaran, Behzad; Movassaghpour, AliAkbar; Farajnia, Safar; Omidi, Yadollah

2013-01-01

Purpose: Recombinant tumor necrosis factor-alpha (TNF-α) has been utilized as an antineoplastic agent for the treatment of patients with melanoma and sarcoma. It targets tumor cell antigens by impressing tumor-associated vessels. Protein purification with affinity chromatography has been widely used in the downstream processing of pharmaceutical-grade proteins. Methods:In this study, we examined the potential of our produced anti-TNF-α scFv fragments for purification of TNF-α produced by Raji cells. The Raji cells were induced by lipopolysaccharides (LPS) to express TNF-α. Western blotting and Fluorescence-activated cell sorting (FACS) flow cytometry analyses were used to evaluate the TNF-α expression. The anti-TNF-α scFv selected from antibody phage display library was coupled to CNBr-activated sepharose 4B beads used for affinity purification of expressed TNF-α and the purity of the protein was assessed by SDS-PAGE. Results: Western blot and FACS flow cytometry analyses showed the successful expression of TNF-α with Raji cells. SDS-PAGE analysis showed the performance of scFv for purification of TNF-α protein with purity over 95%. Conclusion: These findings confirm not only the potential of the produced scFv antibody fragments but also this highly pure recombinant TNF-α protein can be applied for various in vitro and in vivo applications. PMID:24312807

Performance analysis and experimental study on rainfall water purification with an extensive green roof matrix layer in Shanghai, China.

PubMed

Guo, Jiankang; Zhang, Yanting; Che, Shengquan

2018-02-01

Current research has validated the purification of rainwater by a substrate layer of green roofs to some extent, though the effects of the substrate layer on rainwater purification have not been adequately quantified. The present study set up nine extensive green roof experiment combinations based on the current conditions of precipitation characteristics observed in Shanghai, China. Different rain with pollutants were simulated, and the orthogonal design L9 (33) test was conducted to measure purification performance. The purification influences of the extensive green roof substrate layer were quantitatively analyzed in Shanghai to optimize the thickness, proportion of substrate, and sodium polyacrylate content. The experimental outcomes resulted in ammonium nitrogen (NH 4 + -N), lead (Pb), and zinc (Zn) removal of up to 93.87%, 98.81%, and 94.55% in the artificial rainfall, respectively, and NH 4 + -N, Pb, and Zn event mean concentration (EMC) was depressed to 0.263 mg/L, 0.002 mg/L and 0.018 mg/L, respectively, which were all well below the pollutant concentrations of artificial rainfall. With reference to the rainfall chemical characteristics of Shanghai, a combination of a 200 mm thickness, proportions of 1:1:2 of Loam: Perlite: Cocopeat and 2 g/L sodium polyacrylate content was suggested for the design of an extensive green roof substrate to purify NH 4 + -N, Pb and Zn.

[Studies on technology optimization for extraction and purification of total flavones from root bark of Artocarpus styracifolius].

PubMed

Ren, Gang; Liu, Rong-hua; Shao, Feng; Huang, Hui-lian; Wen, Li-rong

2010-08-01

To study the technology optimization for extraction and purification of total flavones from root bark of Artocarpus styracifolius. The optimum extraction conditions were investigated by the contents of the total flavones, using orthogonal test; Static adsorption capacity and desorption rate were employed as examine items for the screening of optimum macroporous resin and optimum technology for the purification of total flavones with selected macroporous were also investigated. The optimum extraction conditions were as follows: using 60% alcohol of seven times than amounts of original material soaking 12 hours,extracting once with hot reflux method at 50 degrees C. HPD-500 type macroporous resin showed better adsorption and desorption property. The optimum purification conditions were as follows: the sample solution was prepared at the concentration of 50.0 mg/mL, subjected to HPD-500 type macroporous resin column chromatography with a load ratio of 22.0 mg total flavones per gram of resin. After standing for 1 hour, the column was eluted with 4 BV water before being eluted with 4 BV 80% alcohol. The purity of the product was 86.4%, which enhanced the content of total flavones by 533%. The optimum conditions for extraction and purification of total flavones from root bark of Artocarpus styractifolius are convenient and practical, and could be used as a reference for industrial production.

Purification of inclusion bodies using PEG precipitation under denaturing conditions to produce recombinant therapeutic proteins from Escherichia coli.

PubMed

Chen, Huanhuan; Li, Ninghuan; Xie, Yueqing; Jiang, Hua; Yang, Xiaoyi; Cagliero, Cedric; Shi, Siwei; Zhu, Chencen; Luo, Han; Chen, Junsheng; Zhang, Lei; Zhao, Menglin; Feng, Lei; Lu, Huili; Zhu, Jianwei

2017-07-01

It has been documented that the purification of inclusion bodies from Escherichia coli by size exclusion chromatography (SEC) may benefit subsequent refolding and recovery of recombinant proteins. However, loading volume and the high cost of the column limits its application in large-scale manufacturing of biopharmaceutical proteins. We report a novel process using polyethylene glycol (PEG) precipitation under denaturing conditions to replace SEC for rapid purification of inclusion bodies containing recombinant therapeutic proteins. Using recombinant human interleukin 15 (rhIL-15) as an example, inclusion bodies of rhIL-15 were solubilized in 7 M guanidine hydrochloride, and rhIL-15 was precipitated by the addition of PEG 6000. A final concentration of 5% (w/v) PEG 6000 was found to be optimal to precipitate target proteins and enhance recovery and purity. Compared to the previously reported S-200 size exclusion purification method, PEG precipitation was easier to scale up and achieved the same protein yields and quality of the product. PEG precipitation also reduced manufacturing time by about 50 and 95% of material costs. After refolding and further purification, the rhIL-15 product was highly pure and demonstrated a comparable bioactivity with a rhIL-15 reference standard. Our studies demonstrated that PEG precipitation of inclusion bodies under denaturing conditions holds significant potential as a manufacturing process for biopharmaceuticals from E. coli protein expression systems.

Purification of Houttuynia cordata Thunb. Essential Oil Using Macroporous Resin Followed by Microemulsion Encapsulation to Improve Its Safety and Antiviral Activity.

PubMed

Pang, Jianmei; Dong, Wujun; Li, Yuhuan; Xia, Xuejun; Liu, Zhihua; Hao, Huazhen; Jiang, Lingmin; Liu, Yuling

2017-02-15

Essential oil extracted from Houttuynia cordata Thunb. ( H. cordata ) is widely used in traditional Chinese medicine due to its excellent biological activities. However, impurities and deficient preparations of the essential oil limit its safety and effectiveness. Herein, we proposed a strategy to prepare H. cordata essential oil (HEO) safely and effectively by combining the solvent extraction and the macroporous resin purification flexibly, and then encapsulating it using microemulsion. The extraction and purification process were optimized by orthogonal experimental design and adsorption-desorption tests, respectively. The average houttuynin content in pure HEO was then validated at 44.3% ± 2.01%, which presented a great potential for industrial application. Subsequently, pure HEO-loaded microemulsion was prepared by high-pressure homogenization and was then fully characterized. Results showed that the pure HEO-loaded microemulsion was successfully prepared with an average particle size of 179.1 nm and a high encapsulation rate of 94.7%. Furthermore, safety evaluation tests and in vitro antiviral testing indicated that the safety and activity of HEO were significantly improved after purification using D101 resin and were further improved by microemulsion encapsulation. These results demonstrated that the purification of HEO by macroporous resin followed by microemulsion encapsulation would be a promising approach for industrial application of HEO for the antiviral therapies.

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

PubMed Central

Pua, Teen-Lee; Chan, Xiao Ying; Loh, Hwei-San; Omar, Abdul Rahman; Yusibov, Vidadi; Musiychuk, Konstantin; Hall, Alexandra C.; Coffin, Megan V.; Shoji, Yoko; Chichester, Jessica A.; Bi, Hong; Streatfield, Stephen J.

2017-01-01

ABSTRACT Highly pathogenic avian influenza (HPAI) H5N1 is an ongoing global health concern due to its severe sporadic outbreaks in Asia, Africa and Europe, which poses a potential pandemic threat. The development of safe and cost-effective vaccine candidates for HPAI is considered the best strategy for managing the disease and addressing the pandemic preparedness. The most potential vaccine candidate is the antigenic determinant of influenza A virus, hemagglutinin (HA). The present research was aimed at developing optimized expression in Nicotiana benthamiana and protein purification process for HA from the Malaysian isolate of H5N1 as a vaccine antigen for HPAI H5N1. Expression of HA from the Malaysian isolate of HPAI in N. benthamiana was confirmed, and more soluble protein was expressed as truncated HA, the HA1 domain over the entire ectodomain of HA. Two different purification processes were evaluated for efficiency in terms of purity and yield. Due to the reduced yield, protein degradation and length of the 3-column purification process, the 2-column method was chosen for target purification. Purified HA1 was found immunogenic in mice inducing H5 HA-specific IgG and a hemagglutination inhibition antibody. This paper offers an alternative production system of a vaccine candidate against a locally circulating HPAI, which has a regional significance. PMID:27929750

Affinity purification and mass spectrometry: an attractive choice to investigate protein-protein interactions in plant immunity

USDA-ARS?s Scientific Manuscript database

Affinity purification of protein complexes from biological tissues, followed by liquid chromatography- tandem mass spectrometry (AP-MS/MS), has ballooned in recent years due to sizeable increases in nucleic acid sequence data essential for interpreting mass spectra, improvements in affinity purifica...

The Extraction and Partial Purification of Bacterial DNA as a Practical Exercise for GCE Advanced Level Students.

ERIC Educational Resources Information Center

Falconer, A. C.; Hayes, L. J.

1986-01-01

Describes a relatively simple method of extraction and purification of bacterial DNA. This technique permits advanced secondary-level science students to obtain adequate amounts of DNA from very small pellets of bacteria and to observe some of its polymer properties. (ML)

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

DOEpatents

Mitchell, Alexander R [Livermore, CA; Coburn, Michael D [Santa Fe, NM; Lee, Gregory S [San Ramon, CA; Schmidt, Robert D [Livermore, CA; Pagoria, Philip F [Livermore, CA; Hsu, Peter C [Pleasanton, CA

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.

21 CFR 74.102 - FD&C Blue No. 2.

Code of Federal Regulations, 2010 CFR

2010-04-01

... subjected to purification procedures. The indigo (or indigo paste) used above is manufactured by the fusion... purification procedures prior to sulfonation. (2) Color additive mixtures for food use (including dietary... (calculated as sodium salts), not more than 15 percent. Water insoluble matter, not more than 0.4 percent...

21 CFR 74.102 - FD&C Blue No. 2.

Code of Federal Regulations, 2012 CFR

2012-04-01

... subjected to purification procedures. The indigo (or indigo paste) used above is manufactured by the fusion... purification procedures prior to sulfonation. (2) Color additive mixtures for food use (including dietary... (calculated as sodium salts), not more than 15 percent. Water insoluble matter, not more than 0.4 percent...

TiO2-Based Advanced Oxidation Nanotechnologies For Water Purification And Reuse

EPA Science Inventory

TiO₂ photocatalysis, one of the UV-based advanced oxidation technologies (AOTs) and nanotechnologies (AONs), has attracted great attention for the development of efficient water treatment and purification systems due to the effectiveness of TiO₂ to generate ...

21 CFR 74.102 - FD&C Blue No. 2.

Code of Federal Regulations, 2013 CFR

2013-04-01

... subjected to purification procedures. The indigo (or indigo paste) used above is manufactured by the fusion... purification procedures prior to sulfonation. (2) Color additive mixtures for food use (including dietary... (calculated as sodium salts), not more than 15 percent. Water insoluble matter, not more than 0.4 percent...

21 CFR 74.102 - FD&C Blue No. 2.

Code of Federal Regulations, 2014 CFR

2014-04-01

... subjected to purification procedures. The indigo (or indigo paste) used above is manufactured by the fusion... purification procedures prior to sulfonation. (2) Color additive mixtures for food use (including dietary... (calculated as sodium salts), not more than 15 percent. Water insoluble matter, not more than 0.4 percent...

Development of concept for concurrent biocide generation and water system purification. [with application to Skylab water tanks

NASA Technical Reports Server (NTRS)

1974-01-01

An attempt was made to construct an electrochemical system, using iodine, for water purification in Skylab. Data cover measurements of iodine production rates, effect of electrode size and geometry on iodine production rates, and feasibility of using stainless steels as reference electrodes.

21 CFR 74.102 - FD&C Blue No. 2.

Code of Federal Regulations, 2011 CFR

2011-04-01

... subjected to purification procedures. The indigo (or indigo paste) used above is manufactured by the fusion... purification procedures prior to sulfonation. (2) Color additive mixtures for food use (including dietary... (calculated as sodium salts), not more than 15 percent. Water insoluble matter, not more than 0.4 percent...

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

DTIC Science & Technology

2016-08-01

Solubilization .................................................2  2.4  Denaturing Chromatography (Purification Step 1...Concentration Determination ................................................................4  2.10  Enzyme -Linked Immunosorbent Assay (ELISA...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in

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…

Protocol for Initial Purification of Bacteriocin

DTIC Science & Technology

2015-10-01

lysate/extract preparation, column purification, and a desalting . The peptide was tracked throughout the process using a soft agar overlay activity...tris PAGE. It is necessary to desalt those samples for 150-mM and 1-M fractions, by using dialysis or G10 sephadex columns, in order to prevent

Extraction of neptunium by trilaurylamine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patil, S.K.; Swarup, R.; Ramaniah, M.V.

1972-07-01

Trilaurylamine (TLA) is considered as useful solvent for the final purification of plutonium and neptunium. As TLA is considered as an alternate possible extractant for the final purification of plutonium and neptunium at Tarapur Reprocessing Plant under construction, it was considered necessary to study the optimum conditions for the extraction of neptunium using TLA.

Field Testing of a Small Water Purification System for Non-PRASA Rural Communities

EPA Science Inventory

Small, rural communities typically do not have adequate water purification systems to sustain their life quality and residents are exposed to pathogens present in drinking water. In Puerto Rico (PR), approximately 4% of the population does not have access to drinking water provi...

PURIFICATION OF RAT LEYDIG CELLS: INCREASED YIELDS AFTER UNIT-GRAVITY SEDIMENTATION OF COLLAGENASE-DISPERSED INTERSTITIAL CELLS

EPA Science Inventory

Abstract

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure, that includes a filtration with nylon mesh (100 micron pore size) to separate interstitial cells from the seminiferous tubules, combining centr...

A Practical Introduction to Separation and Purification Techniques for the Beginning Organic Chemistry Laboratory.

ERIC Educational Resources Information Center

Leonard, Jack E.

1981-01-01

Describes a sequence of experiments developed at Texas A&M University for use in one-semester and two-semester (nonmajors) organic chemistry courses to teach a maximum number of separation and purification techniques such as distillations, recrystallization, liquid-liquid extraction, and chromatography. (SK)

Results from KamLAND-Zen

NASA Astrophysics Data System (ADS)

Asakura, K.; Gando, A.; Gando, Y.; Hachiya, T.; Hayashida, S.; Ikeda, H.; Inoue, K.; Ishidoshiro, K.; Ishikawa, T.; Ishio, S.; Koga, M.; Matsuda, R.; Matsuda, S.; Mitsui, T.; Motoki, D.; Nakamura, K.; Obara, S.; Oki, Y.; Otani, M.; Oura, T.; Shimizu, I.; Shirahata, Y.; Shirai, J.; Suzuki, A.; Tachibana, H.; Tamae, K.; Ueshima, K.; Watanabe, H.; Xu, B. D.; Yamauchi, Y.; Yoshida, H.; Kozlov, A.; Takemoto, Y.; Yoshida, S.; Fushimi, K.; Banks, T. I.; Freedman, S. J.; Fujikawa, B. K.; O'Donnell, T.; Winslow, L. A.; Berger, B. E.; Efremenko, Y.; Karwowski, H. J.; Markoff, D. M.; Tornow, W.; Detwiler, J. A.; Enomoto, S.; Decowski, M. P.

2015-07-01

KamLAND-Zen reports on a preliminary search for neutrinoless double-beta decay with 136Xe based on 114.8 live-days after the purification of the xenon loaded liquid scintillator. In this data, the problematic 110mAg background peak identified in previous searches is reduced by more than a factor of 10. By combining the KamLAND-Zen pre- and post-purification data, we obtain a preliminary lower limit on the 0νββ decay half-life of T1/2 0 ν>2.6 ×1025 yr at 90% C.L. The search sensitivity will be enhanced with additional low background data after the purification. Prospects for further improvements with future KamLAND-Zen upgrades are also presented.

Preparative Purification of Recombinant Proteins: Current Status and Future Trends

PubMed Central

Saraswat, Mayank; Ravidá, Alessandra; Holthofer, Harry

2013-01-01

Advances in fermentation technologies have resulted in the production of increased yields of proteins of economic, biopharmaceutical, and medicinal importance. Consequently, there is an absolute requirement for the development of rapid, cost-effective methodologies which facilitate the purification of such products in the absence of contaminants, such as superfluous proteins and endotoxins. Here, we provide a comprehensive overview of a selection of key purification methodologies currently being applied in both academic and industrial settings and discuss how innovative and effective protocols such as aqueous two-phase partitioning, membrane chromatography, and high-performance tangential flow filtration may be applied independently of or in conjunction with more traditional protocols for downstream processing applications. PMID:24455685

Study on the treatment of acute thallium poisoning.

PubMed

Zhang, Hong-Tao; Qiao, Bao-Ping; Liu, Bao-Ping; Zhao, Xian-Guo

2014-05-01

Acute thallium poisoning rarely occurs but is a serious and even fatal medical condition. Currently, patients with acute thallium poisoning are usually treated with Prussian blue and blood purification therapy. However, there are few studies about these treatments for acute thallium poisoning. Nine patients with acute thallium poisoning from 1 family were treated successfully with Prussian blue and different types of blood purification therapies and analyzed. Prussian blue combined with sequential hemodialysis, hemoperfusion and/or continuous veno-venous hemofiltration were effective for the treatment of patients with acute thallium poisoning, even after delayed diagnosis. Blood purification therapies help in the clearance of thallium in those with acute thallium poisoning. Prussian blue treatment may do the benefit during this process.

Expression and Purification of Rat Glucose Transporter 1 in Pichia pastoris.

PubMed

Venskutonytė, Raminta; Elbing, Karin; Lindkvist-Petersson, Karin

2018-01-01

Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.

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

Extraction and purification methods in downstream processing of plant-based recombinant proteins.

PubMed

Łojewska, Ewelina; Kowalczyk, Tomasz; Olejniczak, Szymon; Sakowicz, Tomasz

2016-04-01

During the last two decades, the production of recombinant proteins in plant systems has been receiving increased attention. Currently, proteins are considered as the most important biopharmaceuticals. However, high costs and problems with scaling up the purification and isolation processes make the production of plant-based recombinant proteins a challenging task. This paper presents a summary of the information regarding the downstream processing in plant systems and provides a comprehensible overview of its key steps, such as extraction and purification. To highlight the recent progress, mainly new developments in the downstream technology have been chosen. Furthermore, besides most popular techniques, alternative methods have been described. Copyright © 2015 Elsevier Inc. All rights reserved.

Expression and Purification of a Matrix Metalloprotease Transmembrane Domain in Escherichia coli.

PubMed

Galea, Charles A

2017-01-01

Membrane tethered matrix metalloproteases are bound to the plasma membrane by a glycosylphosphatidylinositol-anchor or a transmembrane domain. To date, most studies of membrane-bound matrix metalloprotease have focused on the globular catalytic and protein-protein interaction domains of these enzymes. However, the transmembrane domains have been poorly studied even though they are known to mediate intracellular signaling via interaction with various cellular proteins. The expression and purification of the transmembrane domain of these proteins can be challenging due to their hydrophobic nature. In this chapter we describe the purification of a transmembrane domain for a membrane-bound matrix metalloprotease expressed in E. coli and its initial characterization by NMR spectroscopy.

Replacing "Them" with "Us": Language Ideologies and Practices of "Purification" on Facebook

ERIC Educational Resources Information Center

Karimzad, Farzad; Sibgatullina, Gulnaz

2018-01-01

Adopting an online ethnographic approach, we examine the linguistic/semiotic practices and ideologies of "purism" among Tatar and Iranian Azerbaijani Facebook users. We argue that purification practices can be understood as identity work, the outcome of which is often not an etymologically "purer" language but a (perceived)…

Experimental purification of single qubits.

PubMed

Ricci, M; De Martini, F; Cerf, N J; Filip, R; Fiurásek, J; Macchiavello, C

2004-10-22

We report the experimental realization of the purification protocol for single qubits sent through a depolarizing channel. The qubits are associated with polarization states of single photons and the protocol is achieved by means of passive linear optical elements. The present approach may represent a convenient alternative to the distillation and error correction protocols of quantum information.

The Matching Criterion Purification for Differential Item Functioning Analyses in a Large-Scale Assessment

ERIC Educational Resources Information Center

Lee, HyeSun; Geisinger, Kurt F.

2016-01-01

The current study investigated the impact of matching criterion purification on the accuracy of differential item functioning (DIF) detection in large-scale assessments. The three matching approaches for DIF analyses (block-level matching, pooled booklet matching, and equated pooled booklet matching) were employed with the Mantel-Haenszel…

Synthesis, Purification, and Characterization of a [mu]-(1,3-Propanedithiolato)-Hexacarbonyldiiron

ERIC Educational Resources Information Center

Works, Carmen F.

2007-01-01

A project which exposes students to biologically important transition-metal chemistry is illustrated by taking an example of the iron-carbonyl compound, [mu]-(1,3-Propanedithiolaro)-hexa-carbonyldiiron as a structural model for an iron-only hydro-genase. The project provides the students with experience of Schlenk line techniques, purification,…

Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification

ERIC Educational Resources Information Center

Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques

2004-01-01

Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course "Introduction to Genetic Engineering." The heterologous expression of the green fluorescent protein (GFP)* of "Aequorea victoria" is an interesting system for didactic purposes because it can be viewed easily during…

78 FR 38078 - Biweekly Notice; Applications and Amendments to Facility Operating Licenses and Combined Licenses...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-06-25

... SFP Purification Loop and recirculation and purification of the RWST water using the BARS is not... revise the minimum volume and low level setpoint on the Refueling Water Storage Tank. Because the... proposed change would revise Technical Specification 3.5.4, ``Refueling Water Storage Tank (RWST)'' such...

Purification and Electrophoretic Characterization of Lactate Dehydrogenase from Mammalian Blood: A Different Twist on a Classic Experiment

ERIC Educational Resources Information Center

Brunauer, Linda S.

2016-01-01

A multiweek protein purification suite, suitable for upper-division biochemistry or biotechnology undergraduate students, is described. Students work in small teams to isolate the enzyme lactate dehydrogenase (LDH) from a nontraditional tissue source, mammalian blood, using a sequence of three column chromatographic procedures: ion-exchange, size…

From Egg to Crystal: A Practical on Purification, Characterization, and Crystallization of Lysozyme for Bachelor Students

ERIC Educational Resources Information Center

Olieric, Vincent; Schreiber, Angelique; Lorber, Bernard; Putz, Joern

2007-01-01

A practical hands-on course encompassing enzyme purification, biochemical characterization, and crystallization that completed the course work of 350 second-year bachelor students enrolled in molecular biology/biochemistry was given at the Universite Louis Pasteur of Strasbourg (France). The experimental part of the practical dealt entirely with…

Scheme for air treatment in welding workshop

NASA Astrophysics Data System (ADS)

Wang, Gang; Wu, Jin

2017-04-01

There are two major ways to control the pollution of welding fume which are respectively local purification and comprehensive purification. In this paper, the practical welding workshop at school is taken as an example to realize fume treatment in different training conditions by adopting the scheme in which two major ways are combined.

Evaluation of BAUER K-20 Diesel Powered High Pressure Breathing Air Compressor and the P-5 Purification System (Unmanned)

DTIC Science & Technology

1991-08-01

sieve and hopcalite using Bauer cartridge No. 068416. The molecular sieve absorbs oil and water vapors. The hopcalite converts carbon monoxide (CO) to...Molecular Sieve (058825)/ Hopcalite (068416) Cartridge purification system Evaluation. 4. MIL-C-52973A(ME) Military Specification Compressor Unit, 20 CFM

One-step purification of nisin A by immunoaffinity chromatography.

PubMed

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-12-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method.

Purification and characterization of xylooligosaccharides (XOS) from Miscanthus x giganteus

USDA-ARS?s Scientific Manuscript database

Our previous investigation showed that xylooligosaccharides (XOS) could be produced effectively from Miscanthus x giganteus (MxG). Using autohydrolysis, an XOS yield of to 13.5% (w/w) of initial biomass and xylan yield of 69.2% (w/w) was observed. In this study, we investigated the purification of X...

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.

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.

24 CFR 203.52 - Acceptance of individual residential water purification equipment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 24 Housing and Urban Development 2 2011-04-01 2011-04-01 false Acceptance of individual residential water purification equipment. 203.52 Section 203.52 Housing and Urban Development Regulations Relating to Housing and Urban Development (Continued) OFFICE OF ASSISTANT SECRETARY FOR HOUSING-FEDERAL HOUSING COMMISSIONER, DEPARTMENT OF HOUSING AND...

Purification of cardiolipin for surface pressure studies.

PubMed

Houle, A; Téchy, F; Aghion, J; Leblanc, R M

1982-03-01

Thin-layer chromatography and surface pressure-area isotherms of commercial bovine cardiolipins showed that the samples contained contaminants. They were purified by TLC and their purity was checked by chromatography and by their monolayer properties. The molecular area of cardiolipin and its purification yield depend upon the fatty acid composition, particularly the degree of unsaturation.

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

Protein purification and crystallization artifacts: The tale usually not told.

PubMed

Niedzialkowska, Ewa; Gasiorowska, Olga; Handing, Katarzyna B; Majorek, Karolina A; Porebski, Przemyslaw J; Shabalin, Ivan G; Zasadzinska, Ewelina; Cymborowski, Marcin; Minor, Wladek

2016-03-01

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building. © 2016 The Protein Society.

Preparative isolation, fast centrifugal partition chromatography purification and biological activity of cajaflavanone from Derris ferruginea stems.

PubMed

Morel, Sylvie; Landreau, Anne; Nguyen, Van Hung; Derbré, Séverine; Grellier, Philippe; Pape, Patrice Le; Pagniez, Fabrice; Litaudon, Marc; Richomme, Pascal

2012-01-01

The Derris genus is known to contain flavonoid derivatives, including prenylated flavanones and isoflavonoids such as rotenoids, which are generally associated with significant biological activity. To develop an efficient preparative isolation procedure for bioactive cajaflavanone. Fast centrifugal partition chromatography (FCPC) was optimised to purify cajaflavanone from Derris ferruginea stems in a single step as compared to fractionation from the cyclohexane extract by successive conventional solid-liquid chromatography procedures. The purification yield, purity, time and solvent consumption per procedure are described. The anti-fungal, anti-bacterial, anti-leishmanial, anti-plasmodial, anti-oxidant activities and the inhibition of advanced glycation end-products (AGEs) by cajaflavanone accumulation are described. FCPC enabled cajaflavanone purification in a single separation step, yielding sufficient quantities to perform in vitro biological screening. Interestingly, cajaflavanone had an inhibitory effect on the formation of AGEs, without displaying any in vitro anti-oxidant activity. A simple and efficient procedure, in comparison with other preparative methods, for bioactive cajaflavone purification has been developed using FCPC. Copyright © 2011 John Wiley & Sons, Ltd.

Functional magnetic mesoporous nanoparticles for efficient purification of laccase from fermentation broth in magnetically stabilized fluidized bed.

PubMed

Wang, Feng; Guo, Chen; Liu, Chun-Zhao

2013-12-01

A magnetically stabilized fluidized bed (MSFB) with the Cu(2+)-chelated magnetic mesoporous silica nanoparticles (MMSNPs-Cu(2+)) was established to purify laccase directly from the fermentation broth of Trametes versicolor. The MMSNPs-Cu(2+) particles in the MSFB maintained a stable bed expansion of two to threefold at a flow rate of 120-180 cm/h. At the optimal magnetic field intensity of 120 Gs, both the maximal Bodenstein number and the smallest axial dispersion coefficient were achieved, which resulted in a stable fluidization stage. The dynamic binding capacity of laccase in the MSFB decreased from 192.5 to144.3 mg/g when the flow velocity through the bed increased from 44.2 to 69.8 cm/h. The MSFB with MMSNPs-Cu(2+) achieved efficient laccase purification from the fermentation broth with 62.4-fold purification of laccase and 108.9 % activity yield. These results provided an excellent platform for the application of these magnetic mesoporous nanoparticles integrated with the MSFB in developing novel protein purification process.

The Toxic Truth About Carbon Nanotubes in Water Purification: a Perspective View.

PubMed

Das, Rasel; Leo, Bey Fen; Murphy, Finbarr

2018-06-18

Without nanosafety guidelines, the long-term sustainability of carbon nanotubes (CNTs) for water purifications is questionable. Current risk measurements of CNTs are overshadowed by uncertainties. New risks associated with CNTs are evolving through different waste water purification routes, and there are knowledge gaps in the risk assessment of CNTs based on their physical properties. Although scientific efforts to design risk estimates are evolving, there remains a paucity of knowledge on the unknown health risks of CNTs. The absence of universal CNT safety guidelines is a specific hindrance. In this paper, we close these gaps and suggested several new risk analysis roots and framework extrapolations from CNT-based water purification technologies. We propose a CNT safety clock that will help assess risk appraisal and management. We suggest that this could form the basis of an acceptable CNT safety guideline. We pay particular emphasis on measuring risks based on CNT physico-chemical properties such as diameter, length, aspect ratio, type, charge, hydrophobicity, functionalities and so on which determine CNT behaviour in waste water treatment plants and subsequent release into the environment.

Electro-purification of carbon nanotube networks without damaging the assembly structure and crystallinity

NASA Astrophysics Data System (ADS)

Yang, Xueqin; Yang, Ming; Zhang, Huichao; Zhao, Jingna; Zhang, Xiaohua; Li, Qingwen

2018-06-01

Fe-containing nanoparticles are of a high mass fraction in the as-grown carbon nanotube (CNT) network. By controlling the S-to-Fe atom ratio in the growth feedstock and introducing water as a weak oxidant, highly crystalline few-walled CNT network can be obtained, with a mass fraction of over 20 wt% for the Fe-containing nanoparticles. We report here an electron-oxidation-based purification method to efficiently remove the Fe-containing nanoparticles without inducing clear damage to either the assembly structure or the tube crystallinity. The purification could increase the ratio between Raman D and G peak intensities slightly from 0.08 to 0.12, decrease the specific conductivity from 0.31 to 0.24 S m2/g and the Fe content from >20 wt% to ≈1 wt%, and modify the capacitance just by about 13 F/g. All these indicate that the CNT network was well maintained by such gentle electro-oxidation-based purification. In addition, the purified CNT network can exhibit advantages in mechanical and electrical applications.

Catalase purification from rat liver with iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) cryogel discs.

PubMed

Göktürk, Ilgım; Perçin, Işık; Denizli, Adil

2016-08-17

In this study, iron-chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(l)-glutamic acid) (PHEMAGA/Fe(3+)) cryogel discs were prepared. The PHEMAGA/Fe(3+) cryogel discs were characterized by elemental analysis, scanning electron microscopy, Fourier transform infrared spectroscopy, swelling tests, and surface area measurements. The PHEMAGA/Fe(3+) cryogel discs had large pores ranging from 10 to 100 µm with a swelling degree of 9.36 g H2O/g cryogel. Effects of pH, temperature, initial catalase concentration, and flow rate on adsorption capacity of the PHEMAGA/Fe(3+) cryogel discs were investigated. Maximum catalase adsorption capacity (62.6 mg/g) was obtained at pH 7.0, 25°C, and 3 mg/ml initial catalase concentration. The PHEMAGA/Fe(3+) cryogel discs were also tested for the purification of catalase from rat liver. After tissue homogenization, purification of catalase was performed using the PHEMAGA/Fe(3+) cryogel discs and catalase was obtained with a yield of 54.34 and 16.67 purification fold.

Calculation of Purification Systems of Hydrocarbonmoderated Reactors; CALCULO DE SISTEMAS DE PURIFICATION DE REACTORES MODERADOS FOR HIDROCARBUROS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Santos, A.A.

1958-01-01

culation of Purification Systems of Hydrocarbonmoderated Reactors). Agustin Alonso Santos. 1958. 23p. As as introduction to the calculation of the purification systems of bydrocarbon-moderated reactors, the effects of heat and radiation on the polyphenols are considered. The chemical, physical, and nuclear properties are tabulated. The formation velocity of the polymers and gases, pyrolysis, effects of heat on the polymer, and the activity accumulated in the moderator ars discussed. The calculation is based on the hypetheses that the radiation catalyzes the formation of polymers, the velocity of the polymerization reaction is constant, the polymer concentration is maintained at a limit whichmore » does not adversely affect the heat transfer properties, the velocity of the separation of polymers in the distillation column is in proportion to their concentration in the hydrocarbon and the pyrolysis causes gaseous products. Formulas are derived expressing the purified flow and the activities accumulated in the distillation residues. The results are applied to the parification system of the Organic Moderated Reactor Experiment (J.S.R.)« less

Characterization, Purification of Poncirin from Edible Citrus Ougan (Citrus reticulate cv. Suavissima) and Its Growth Inhibitory Effect on Human Gastric Cancer Cells SGC-7901

PubMed Central

Zhu, Xiaoyan; Luo, Fenglei; Zheng, Yixiong; Zhang, Jiukai; Huang, Jianzhen; Sun, Chongde; Li, Xian; Chen, Kunsong

2013-01-01

Poncirin is a bitter flavanone glycoside with various biological activities. Poncirin was isolated from four different tissues (flavedo, albedo, segment membrane, and juice sac) of Ougan fruit (Citrus reticulate cv. Suavissima). The highest content of poncirin was found in the albedo of Ougan fruit (1.37 mg/g DW). High speed counter-current chromatography (HSCCC) combined with D101 resin chromatography was utilized for the separation and purification of poncirin from the albedo of Ougan fruit. After this two-step purification, poncirin purity increased from 0.14% to 96.56%. The chemical structure of the purified poncirin was identified by both HPLC-PDA and LC-MS. Poncirin showed a significant in vitro inhibitory effect on the growth of the human gastric cancer cells, SGC-7901, in a dose-dependent manner. Thus, poncirin from Ougan fruit, may be beneficial for gastric cancer prevention. The purification method demonstrated here will be useful for further studies on the pharmacological mechanism of poncirin activity, as well as for guiding the consumption of Ougan fruit. PMID:23615464

Biofilm bacterial communities in urban drinking water distribution systems transporting waters with different purification strategies.

PubMed

Wu, Huiting; Zhang, Jingxu; Mi, Zilong; Xie, Shuguang; Chen, Chao; Zhang, Xiaojian

2015-02-01

Biofilm formation in drinking water distribution systems (DWDS) has many adverse consequences. Knowledge of microbial community structure of DWDS biofilm can aid in the design of an effective control strategy. However, biofilm bacterial community in real DWDS and the impact of drinking water purification strategy remain unclear. The present study investigated the composition and diversity of biofilm bacterial community in real DWDSs transporting waters with different purification strategies (conventional treatment and integrated treatment). High-throughput Illumina MiSeq sequencing analysis illustrated a large shift in the diversity and structure of biofilm bacterial community in real DWDS. Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, Nitrospirae, and Cyanobacteria were the major components of biofilm bacterial community. Proteobacteria (mainly Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) predominated in each DWDS biofilm, but the compositions of the dominant proteobacterial classes and genera and their proportions varied among biofilm samples. Drinking water purification strategy could shape DWDS biofilm bacterial community. Moreover, Pearson's correlation analysis indicated that Actinobacteria was positively correlated with the levels of total alkalinity and dissolved organic carbon in tap water, while Firmicutes had a significant positive correlation with nitrite nitrogen.

Purification of bone morphogenetic protein-2 from refolding mixtures using mixed-mode membrane chromatography.

PubMed

Gieseler, Gesa; Pepelanova, Iliyana; Stuckenberg, Lena; Villain, Louis; Nölle, Volker; Odenthal, Uwe; Beutel, Sascha; Rinas, Ursula; Scheper, Thomas

2017-01-01

In this study, we present the development of a process for the purification of recombinant human bone morphogenetic protein-2 (rhBMP-2) using mixed-mode membrane chromatography. RhBMP-2 was produced as inclusion bodies in Escherichia coli. In vitro refolding using rapid dilution was carried out according to a previously established protocol. Different membrane chromatography phases were analyzed for their ability to purify BMP-2. A membrane phase with salt-tolerant properties resulting from mixed-mode ligand chemistry was able to selectively purify BMP-2 dimer from refolding mixtures. No further purification or polishing steps were necessary and high product purity was obtained. The produced BMP-2 exhibited a biological activity of 7.4 × 10 5  U/mg, comparable to commercial preparations. Mixed-mode membrane chromatography can be a valuable tool for the direct purification of proteins from solutions with high-conductivity, for example refolding buffers. In addition, in this particular case, it allowed us to circumvent the use of heparin-affinity chromatography, thus allowing the design of an animal-component-free process.

Choices of capture chromatography technology in antibody manufacturing processes.

PubMed

DiLeo, Michael; Ley, Arthur; Nixon, Andrew E; Chen, Jie

2017-11-15

The capture process employed in monoclonal antibody downstream purification is not only the most critically impacted process by increased antibody titer resulting from optimized mammalian cell culture expression systems, but also the most important purification step in determining overall process throughput, product quality, and economics. Advances in separation technology for capturing antibodies from complex feedstocks have been one focus of downstream purification process innovation for past 10 years. In this study, we evaluated new generation chromatography resins used in the antibody capture process including Protein A, cation exchange, and mixed mode chromatography to address the benefits and unique challenges posed by each chromatography approach. Our results demonstrate the benefit of improved binding capacity of new generation Protein A resins, address the concern of high concentration surge caused aggregation when using new generation cation exchange resins with over 100mg/mL binding capacity, and highlight the potential of multimodal cation exchange resins for capture process design. The new landscape of capture chromatography technologies provides options to achieve overall downstream purification outcome with high product quality and process efficiency. Copyright © 2017 Elsevier B.V. All rights reserved.

Two-step purification of His-tagged Nef protein in native condition using heparin and immobilized metal ion affinity chromatographies.

PubMed

Finzi, Andrés; Cloutier, Jonathan; Cohen, Eric A

2003-07-01

The Nef protein encoded by human immunodeficiency virus type 1 (HIV-1) has been shown to be an important factor of progression of viral growth and pathogenesis in both in vitro and in vivo. The lack of a simple procedure to purify Nef in its native conformation has limited molecular studies on Nef function. A two-step procedure that includes heparin and immobilized metal ion affinity chromatographies (IMACs) was developed to purify His-tagged Nef (His(6)-Nef) expressed in bacteria in native condition. During the elaboration of this purification procedure, we identified two closely SDS-PAGE-migrating contaminating bacterial proteins, SlyD and GCHI, that co-eluted with His(6)-Nef in IMAC in denaturing condition and developed purification steps to eliminate these contaminants in native condition. Overall, this study describes a protocol that allows rapid purification of His(6)-Nef protein expressed in bacteria in native condition and that removes metal affinity resin-binding bacterial proteins that can contaminate recombinant His-tagged protein preparation.

Gram-scale purification of aconitine and identification of lappaconitine in Aconitum karacolicum.

PubMed

Tarbe, M; de Pomyers, H; Mugnier, L; Bertin, D; Ibragimov, T; Gigmes, D; Mabrouk, K

2017-07-01

Aconitum karacolicum from northern Kyrgyzstan (Alatau area) contains about 0.8-1% aconitine as well as other aconite derivatives that have already been identified. In this paper, we compare several methods for the further purification of an Aconitum karacolicum extract initially containing 80% of aconitine. Reverse-phase flash chromatography, reverse-phase semi-preparative HPLC, centrifugal partition chromatography (CPC) and recrystallization techniques were evaluated regarding first their efficiency to get the highest purity of aconitine (over 96%) and secondly their applicability in a semi-industrial scale purification process (in our case, 150g of plant extract). Even if the CPC technique shows the highest purification yield (63%), the recrystallization remains the method of choice to purify a large amount of aconitine as i) it can be easily carried out in safe conditions; ii) an aprotic solvent is used, avoiding aconitine degradation. Moreover, this study led us to the identification of lappaconitine in Aconitum karacolicum, a well-known alkaloid never found in this Aconitum species. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

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.

Single step purification of recombinant proteins using the metal ion-inducible autocleavage (MIIA) domain as linker for tag removal.

PubMed

Ibe, Susan; Schirrmeister, Jana; Zehner, Susanne

2015-08-20

For fast and easy purification, proteins are typically fused with an affinity tag, which often needs to be removed after purification. Here, we present a method for the removal of the affinity tag from the target protein in a single step protocol. The protein VIC_001052 of the coral pathogen Vibrio coralliilyticus ATCC BAA-450 contains a metal ion-inducible autocatalytic cleavage (MIIA) domain. Its coding sequence was inserted into an expression vector for the production of recombinant fusion proteins. Following, the target proteins MalE and mCherry were produced as MIIA-Strep fusion proteins in Escherichia coli. The target proteins could be separated from the MIIA-Strep part simply by the addition of calcium or manganese(II) ions within minutes. The cleavage is not affected in the pH range from 5.0 to 9.0 or at low temperatures (6°C). Autocleavage was also observed with immobilized protein on an affinity column. The protein yield was similar to that achieved with a conventional purification protocol. Copyright © 2015 Elsevier B.V. All rights reserved.

Fluxing purification and its effect on magnetic properties of high-Bs FeBPSiC amorphous alloy

NASA Astrophysics Data System (ADS)

Pang, Jing; Wang, Anding; Yue, Shiqiang; Kong, Fengyu; Qiu, Keqiang; Chang, Chuntao; Wang, Xinmin; Liu, Chain-Tsuan

2017-07-01

A high-Bs amorphous alloy with the base composition Fe83B11P3Si2C1 was used to study the effects of fluxing purification on amorphous forming ability and magnetic properties of the alloy prepared with raw materials in industrialization. By using fluxing purification, the surface crystallization was suppressed and fully amorphous Fe83B11P3Si2C1 ribbons with a maximum thickness of 48 μm were successfully achieved by using an industrial process and materials. The amorphous ribbons made with industrial-purified alloys exhibit excellent magnetic properties, containing high-Bs of 1.65 T, low Hc of 2.0 A/m, and high μe of 9.7 × 103 at 1 kHz. Impurities in the melting alloys exist in three forms and have different effluences on magnetic properties. The surface crystallization was triggered by the impurities which exist as high melting point inclusions serving as nuclei. Thus, fluxing purification is a feasible way for industrialization of high-Bs FeBPSiC amorphous alloys.

Sophisticated Cloning, Fermentation, and Purification Technologies for an Enhanced Therapeutic Protein Production: A Review

PubMed Central

Gupta, Sanjeev K.; Shukla, Pratyoosh

2017-01-01

The protein productions strategies are crucial towards the development of application based research and elucidating the novel purification strategies for industrial production. Currently, there are few innovative avenues are studies for cloning, upstream, and purification through efficient bioprocess development. Such strategies are beneficial for industries as well as proven to be vital for effectual therapeutic protein development. Though, these techniques are well documented, but, there is scope of addition to current knowledge with novel and new approaches and it will pave new avenues in production of recombinant microbial and non-microbial proteins including secondary metabolites. In this review, we have focussed on the recent development in clone selection, various modern fermentation and purification technologies and future directions in these emerging areas. Moreover, we have also highlighted notable perspectives and challenges involved in the bioengineering of such proteins, including quality by design, gene editing and pioneering ideas. The biopharmaceutical industries continue to shift towards more flexible, automated platforms and economical product development, which in turn can help in developing the cost effective processes and affordable drug development for a large community. PMID:28725194

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. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Experimental entanglement purification of arbitrary unknown states.

PubMed

Pan, Jian-Wei; Gasparoni, Sara; Ursin, Rupert; Weihs, Gregor; Zeilinger, Anton

2003-05-22

Distribution of entangled states between distant locations is essential for quantum communication over large distances. But owing to unavoidable decoherence in the quantum communication channel, the quality of entangled states generally decreases exponentially with the channel length. Entanglement purification--a way to extract a subset of states of high entanglement and high purity from a large set of less entangled states--is thus needed to overcome decoherence. Besides its important application in quantum communication, entanglement purification also plays a crucial role in error correction for quantum computation, because it can significantly increase the quality of logic operations between different qubits. Here we demonstrate entanglement purification for general mixed states of polarization-entangled photons using only linear optics. Typically, one photon pair of fidelity 92% could be obtained from two pairs, each of fidelity 75%. In our experiments, decoherence is overcome to the extent that the technique would achieve tolerable error rates for quantum repeaters in long-distance quantum communication. Our results also imply that the requirement of high-accuracy logic operations in fault-tolerant quantum computation can be considerably relaxed.

Kevlar based nanofibrous particles as robust, effective and recyclable absorbents for water purification.

PubMed

Nie, Chuanxiong; Peng, Zihang; Yang, Ye; Cheng, Chong; Ma, Lang; Zhao, Changsheng

2016-11-15

Developing robust and recyclable absorbents for water purification is of great demand to control water pollution and to provide sustainable water resources. Herein, for the first time, we reported the fabrication of Kevlar nanofiber (KNF) based composite particles for water purification. Both the KNF and KNF-carbon nanotube composite particles can be produced in large-scale by automatic injection of casting solution into ethanol. The resulted nanofibrous particles showed high adsorption capacities towards various pollutants, including metal ions, phenylic compounds and various dyes. Meanwhile, the adsorption process towards dyes was found to fit well with the pseudo-second-order model, while the adsorption speed was controlled by intraparticle diffusion. Furthermore, the adsorption capacities of the nanofibrous particles could be easily recovered by washing with ethanol. In general, the KNF based particles integrate the advantages of easy production, robust and effective adsorption performances, as well as good recyclability, which can be used as robust absorbents to remove toxic molecules and forward the application of absorbents in water purification. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimization of Serine Protease Purification from Mango (Mangifera indica cv. Chokanan) Peel in Polyethylene Glycol/Dextran Aqueous Two Phase System

PubMed Central

Mehrnoush, Amid; Mustafa, Shuhaimi; Sarker, Md. Zaidul Islam; Yazid, Abdul Manap Mohd

2012-01-01

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000–12,000 g·mol−1), tie line length (−3.42–35.27%), NaCl (−2.5–11.5%) and pH (4.5–10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol−1 of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing. PMID:22489172

Optimization of serine protease purification from mango (Mangifera indica cv. Chokanan) peel in polyethylene glycol/dextran aqueous two phase system.

PubMed

Mehrnoush, Amid; Mustafa, Shuhaimi; Sarker, Md Zaidul Islam; Yazid, Abdul Manap Mohd

2012-01-01

Mango peel is a good source of protease but remains an industrial waste. This study focuses on the optimization of polyethylene glycol (PEG)/dextran-based aqueous two-phase system (ATPS) to purify serine protease from mango peel. The activity of serine protease in different phase systems was studied and then the possible relationship between the purification variables, namely polyethylene glycol molecular weight (PEG, 4000-12,000 g·mol(-1)), tie line length (-3.42-35.27%), NaCl (-2.5-11.5%) and pH (4.5-10.5) on the enzymatic properties of purified enzyme was investigated. The most significant effect of PEG was on the efficiency of serine protease purification. Also, there was a significant increase in the partition coefficient with the addition of 4.5% of NaCl to the system. This could be due to the high hydrophobicity of serine protease compared to protein contaminates. The optimum conditions to achieve high partition coefficient (84.2) purification factor (14.37) and yield (97.3%) of serine protease were obtained in the presence of 8000 g·mol(-1) of PEG, 17.2% of tie line length and 4.5% of NaCl at pH 7.5. The enzymatic properties of purified serine protease using PEG/dextran ATPS showed that the enzyme could be purified at a high purification factor and yield with easy scale-up and fast processing.

Aptamer facilitated purification of functional proteins.

PubMed

Beloborodov, Stanislav S; Bao, Jiayin; Krylova, Svetlana M; Shala-Lawrence, Agnesa; Johnson, Philip E; Krylov, Sergey N

2018-01-15

DNA aptamers are attractive capture probes for affinity chromatography since, in contrast to antibodies, they can be chemically synthesized and, in contrast to tag-specific capture probes (such as Nickel-NTA or Glutathione), they can be used for purification of proteins free of genetic modifications (such as His or GST tags). Despite these attractive features of aptamers as capture probes, there are only a few reports on aptamer-based protein purification and none of them includes a test of the purified protein's activity, thus, leaving discouraging doubts about method's ability to purify proteins in their active state. The goal of this work was to prove that aptamers could facilitate isolation of active proteins. We refined a complete aptamer-based affinity purification procedure, which takes 4 h to complete. We further applied this procedure to purify two recombinant proteins, MutS and AlkB, from bacterial cell culture: 0.21 mg of 85%-pure AlkB from 4 mL of culture and 0.24 mg of 82%-pure MutS from 0.5 mL of culture. Finally, we proved protein activity by two capillary electrophoresis based assays: an enzymatic assay for AlkB and a DNA-binding assay for MutS. We suggest that in combination with aptamer selection for non-purified protein targets in crude cell lysate, aptamer-based purification provides a means of fast isolation of tag-free recombinant proteins in their native state without the use of antibodies. Copyright © 2017 Elsevier B.V. All rights reserved.

Large-scale purification and characterization of recombinant human stem cell factor in Escherichia coli.

PubMed

Chen, Liang-Hua; Cai, Feng; Zhang, Dan-Ju; Zhang, Li; Zhu, Peng; Gao, Shun

2017-07-01

The pharmacological importance of recombinant human stem cell factor (rhSCF) has increased the demand to establish effective and large-scale production and purification processes. A good source of bioactive recombinant protein with capability of being scaled-up without losing activity has always been a challenge. The objectives of the study were the rapid and efficient pilot-scale expression and purification of rhSCF. The gene encoding stem cell factor (SCF) was cloned into pBV220 and transformed into Escherichia coli. The recombinant SCF was expressed and isolated using a procedure consisting of isolation of inclusion bodies (IBs), denaturation, and refolding followed by chromatographic steps toward purification. The yield of rhSCF reached 835.6 g/20 L, and the expression levels of rhSCF were about 33.9% of the total E. coli protein content. rhSCF was purified by isolation of IBs, denaturation, and refolding, followed by SP-Sepharose chromatography, Source 30 reversed-phase chromatography, and Q-Sepharose chromatography. This procedure was developed to isolate 5.5 g of rhSCF (99.5% purity) with specific activity at 0.96 × 10 6  IU/mg, endotoxin levels of pyrogen at 1.0 EU/mg, and bacterial DNA at 10 ng/mg. Pilot-scale fermentations and purifications were set up for the production of rhSCF that can be upscaled for industry. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira.

PubMed

Hauk, P; Carvalho, E; Ho, P L

2011-04-01

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Simultaneous separation and purification of flavonoids and oleuropein from Olea europaea L. (olive) leaves using macroporous resin.

PubMed

Li, Chen; Zheng, Yuanyuan; Wang, Xiaofei; Feng, Shilan; Di, Duolong

2011-12-01

This study developed a feasible process to simultaneously separate and purify polyphenols, including flavonoids and oleuropein, from the leaves of Olea europaea L. Macroporous resins were used as the separation and purification materials. The performance and separation capabilities of eight resins (D101, DM130, HPD450, LSA-21, LSA-40, 07C, LSD001 and HPD600) were systematically evaluated. The contents of target polyphenols in different extracts were determined using ultraviolet (for flavonoids) and high-performance liquid chromatographic (for oleuropein) methods. The static adsorption and desorption results showed that resin LSA-21 had better adsorption properties among the eight resins. Influential factors such as extraction method, pH value of feeding solution, desorption solution, adsorption kinetics and adsorption isotherm, etc. to the extraction and purification of these polyphenols were successively investigated on resin LSA-21. The target flavonoids and oleuropein were selectively purified using resin LSA-21. Compared with the contents in raw leaves, the contents of total flavonoids and oleuropein in the final purified products were increased 13.2-fold (from 16 to 211 g kg(-1) ) and 7.5-fold (from 120 to 902 g kg(-1) ) with recovery yields of 87.9% and 85.6%, respectively. This extraction and purification method could be used in the large-scale enrichment or purification of flavonoids, oleuropein and other polyphenols from O. europaea L. leaves or other herbal materials in industrial, food processing and medical manufacture. Copyright © 2011 Society of Chemical Industry.

Use of naturally growing aquatic plants for wastewater purification.

PubMed

Zimmels, Y; Kirzhner, F; Roitman, S

2004-01-01

This paper examines potential uses of naturally growing aquatic plants for wastewater purification. These plants enhance the removal of pollutants by consuming part of them in the form of plant nutrients. This applies to urban and agricultural wastewater, in particular, where treatment units of different sizes can be applied at the pollution source. The effectiveness of wastewater purification by different plants was tested on laboratory and pilot scales. The growth rate of the plants was related to the wastewater content in the water. Batch and semicontinuous experiments verified that the plants are capable of decreasing all tested indicators for water quality to levels that permit the use of the purified water for irrigation. This applies to biochemical oxygen demand (BOD), chemical oxygen demand, total suspended solids. pH, and turbidity. In specific cases, the turbidity reached the level of drinking water. Comparison of BOD concentrations with typical levels in water treatment facilities across the country indicates the effectiveness of water purification with plants. A major effect of treatment with plants was elimination of the disturbing smell from the wastewater. It is shown that mixtures of wastewater and polluted water from the Kishon River are amenable in varying degrees to treatment by the plants. The higher the wastewater content in the mixture, the more effective the treatment by the plants. In this context, a scheme for rehabilitation and restoration of the Kishon River is presented and technical and economical aspects of the purification technology are considered.

Quantitative comparison of structure and dynamics of elastin following three isolation schemes by 13C solid state NMR and MALDI mass spectrometry.

PubMed

Papaioannou, A; Louis, M; Dhital, B; Ho, H P; Chang, E J; Boutis, G S

2015-05-01

Methods for isolating elastin from fat, collagen, and muscle, commonly used in the design of artificial elastin based biomaterials, rely on exposing tissue to harsh pH levels and temperatures that usually denature many proteins. At present, a quantitative measurement of the modifications to elastin following isolation from other extracellular matrix constituents has not been reported. Using magic angle spinning (13)C NMR spectroscopy and relaxation methodologies, we have measured the modification in structure and dynamics following three known purification protocols. Our experimental data reveal that the (13)C spectra of the hydrated samples appear remarkably similar across the various purification methods. Subtle differences in the half maximum widths were observed in the backbone carbonyl suggesting possible structural heterogeneity across the different methods of purification. Additionally, small differences in the relative signal intensities were observed between purified samples. Lyophilizing the samples results in a reduction of backbone motion and reveals additional differences across the purification methods studied. These differences were most notable in the alanine motifs indicating possible changes in cross-linking or structural rigidity. The measured correlation times of glycine and proline moieties are observed to also vary considerably across the different purification methods, which may be related to peptide bond cleavage. Lastly, the relative concentration of desmosine cross-links in the samples quantified by MALDI mass spectrometry is reported. Copyright © 2015 Elsevier B.V. All rights reserved.

Cell purification: a new challenge for biobanks.

PubMed

Almeida, Maria; García-Montero, Andres C; Orfao, Alberto

2014-01-01

Performing '-omics' analyses on heterogeneous biological tissue samples, such as blood or bone marrow, can lead to biased or even erroneous results, particularly when the targeted cells and/or molecules are present at relatively low percentages/amounts. In such cases, whole sample analysis will most probably dilute and mask the features of the cell and/or molecules of interest, and this will negatively impact the results and their interpretation. Therefore, frequently it is critically important to have well-characterized and high-quality purified cell populations for the reliable detection of subtle variations in their specific features, such as gene expression profile, protein expression pattern and metabolic status. Biobanks are technological platforms which aim to provide researchers access to a large number of high-quality biological samples and their associated data, particularly to support high-quality scientific and clinical research projects, and such projects will benefit enormously by having access to high-quality purified cell populations or their biological components (e.g. DNA, RNA, proteins). Therefore, a clear opportunity exists for preparative cell sorting techniques in biobanks. Although multiple different cell purification approaches exist or are under development (e.g. cell purification techniques based on cell adherence, density and/or cell size properties, methods based on antibody binding as well as new lab-on-a-chip purification techniques), the choice for a specific technology depends on multiple variables, including cell recovery, purity and yield, among others. In addition, most cell purification approaches are not well suited for high-throughput (HT) purification of multiple cell populations coexisting in a sample. Here we review the most (currently) used cell sorting methods that may be applied for sample preparation in biobanks. For the different approaches, technical considerations about their advantages and limitations are highlighted, and the requirements to be met by a HT cell sorting technology to be used in biobanks are also discussed.

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 considered as a new phage purification method, appropriate for further investigations and development. PMID:21627821

Profiling and quantitative evaluation of three Nickel-Coated magnetic matrices for purification of recombinant proteins: lelpful hints for the optimized nanomagnetisable matrix preparation

PubMed Central

2011-01-01

Background Several materials are available in the market that work on the principle of protein magnetic fishing by their histidine (His) tags. Little information is available on their performance and it is often quoted that greatly improved purification of histidine-tagged proteins from crude extracts could be achieved. While some commercial magnetic matrices could be used successfully for purification of several His-tagged proteins, there are some which have been proved to operate just for a few extent of His-tagged proteins. Here, we address quantitative evaluation of three commercially available Nickel nanomagnetic beads for purification of two His-tagged proteins expressed in Escherichia coli and present helpful hints for optimized purification of such proteins and preparation of nanomagnetisable matrices. Results Marked differences in the performance of nanomagnetic matrices, principally on the basis of their specific binding capacity, recovery profile, the amount of imidazole needed for protein elution and the extent of target protein loss and purity were obtained. Based on the aforesaid criteria, one of these materials featured the best purification results (SiMAG/N-NTA/Nickel) for both proteins at the concentration of 4 mg/ml, while the other two (SiMAC-Nickel and SiMAG/CS-NTA/Nickel) did not work well with respect to specific binding capacity and recovery profile. Conclusions Taken together, functionality of different types of nanomagnetic matrices vary considerably. This variability may not only be dependent upon the structure and surface chemistry of the matrix which in turn determine the affinity of interaction, but, is also influenced to a lesser extent by the physical properties of the protein itself. Although the results of the present study may not be fully applied for all nanomagnetic matrices, but provide a framework which could be used to profiling and quantitative evaluation of other magnetisable matrices and also provide helpful hints for those researchers facing same challenge. PMID:21824404

Purification and Characterization of a Mucin Specific Mycelial Lectin from Aspergillus gorakhpurensis: Application for Mitogenic and Antimicrobial Activity

PubMed Central

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Background Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Methods Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Results Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5–9.5, while optimum temperature for lectin activity was 20–30°C. Lectin was stable within a pH range of 7.0–10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. Conclusion This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin. PMID:25286160

Purification and characterization of a mucin specific mycelial lectin from Aspergillus gorakhpurensis: application for mitogenic and antimicrobial activity.

PubMed

Singh, Ram Sarup; Kaur, Hemant Preet; Singh, Jatinder

2014-01-01

Lectins are carbohydrate binding proteins or glycoproteins that bind reversibly to specific carbohydrates present on the apposing cells, which are responsible for their ability to agglutinate red blood cells, lymphocytes, fibroblasts, etc. Interest in lectins has been intensified due to their carbohydrate specificity as they can be valuable reagents for the investigation of cell surface sugars, purification and characterization of glycoproteins. The present study reports the purification, characterization and evaluation of mitogenic and antimicrobial potential of a mycelial lectin from Aspergillus gorakhpurensis. Affinity chromatography on mucin-sepharose column was carried out for purification of Aspergillus gorakhpurensis lectin. The lectin was characterized for physico-chemical parameters. Mitogenic potential of the lectin was evaluated against splenocytes of Swiss albino mice by MTT assay. Antimicrobial activity of the purified lectin has also been evaluated by disc diffusion assay. Single-step affinity purification resulted in 18.6-fold purification of the mycelial lectin. The molecular mass of the lectin was found to be 70 kDa and it was composed of two subunits of 34.8 kDa as determined by gel filtration chromatography, SDS-PAGE and MALDI-TOF analysis. pH optima of the lectin was found to be 6.5-9.5, while optimum temperature for lectin activity was 20-30 °C. Lectin was stable within a pH range of 7.0-10.5 and showed fair thermostability. EDTA did not affect lectin activity whereas it was found susceptible to the denaturants tested. MTT assay revealed strong mitogenic potential of A. gorakhpurensis lectin at a concentration upto 150 µg/mL. Antimicrobial activity assay showed its potent antibacterial activity against Bacillus cereus, Staphylococcous aureus and Escherichia coli and marginal antifungal activity against Saccharomyces cerevisiae. This is the first report on the mitogenic and antimicrobial potential of Aspergillus gorakhpurensis lectin. The results will provide useful guidelines for further research in clinical applications of this lectin.

Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein.

PubMed

Ding, Xueqin; Wang, Yuzhi; Zeng, Qun; Chen, Jing; Huang, Yanhua; Xu, Kaijia

2014-03-07

A series of novel cationic functional hexaalkylguanidinium ionic liquids and anionic functional tetraalkylguanidinium ionic liquids have been devised and synthesized based on 1,1,3,3-tetramethylguanidine. The structures of the ionic liquids (ILs) were confirmed by (1)H nuclear magnetic resonance ((1)H NMR) and 13C nuclear magnetic resonance (13C NMR) and the production yields were all above 90%. Functional guanidinium ionic liquid aqueous two-phase systems (FGIL-ATPSs) have been first designed with these functional guanidinium ILs and phosphate solution for the purification of protein. After phase separation, proteins had transferred into the IL-rich phase and the concentrations of proteins were determined by measuring the absorbance at 278 nm using an ultra violet visible (UV-vis) spectrophotometer. The advantages of FGIL-ATPSs were compared with ordinary ionic liquid aqueous two-phase systems (IL-ATPSs). The proposed FGIL-ATPS has been applied to purify lysozyme, trypsin, ovalbumin and bovine serum albumin. Single factor experiments were used to research the effects of the process, such as the amount of ionic liquid (IL), the concentration of salt solution, temperature and the amount of protein. The purification efficiency reaches to 97.05%. The secondary structure of protein during the experimental process was observed upon investigation using UV-vis spectrophotometer, Fourier-transform infrared spectroscopy (FT-IR) and circular dichroism spectrum (CD spectrum). The precision, stability and repeatability of the process were investigated. The mechanisms of purification were researched by dynamic light scattering (DLS), determination of the conductivity and transmission electron microscopy (TEM). It was suggested that aggregation and embrace phenomenon play a significant role in the purification of proteins. All the results show that FGIL-ATPSs have huge potential to offer new possibility in the purification of proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

ERIC Educational Resources Information Center

Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

2008-01-01

We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

Purification of NAD glycohydrolase from Agkistrodon acutus venom.

PubMed

Wu, Shuang Ding; Liu, Yanli; Xu, Xiaolong; Zhu, Zhengang

2002-07-01

NAD glycohydrolase (NADase) from Agkistrodon acutus venom was purified to electrophoretic homogeneity by a fast, reproducible 3-step procedure including Q Sepharose Fast Flow, Superdex 75, and Mono S column chromatography. This new procedure gave a 15.6-fold purification with a recovery yield of 7.9% and a specific activity of 12.8 units/mg.

Designing and Conducting a Purification Scheme as an Organic Chemistry Laboratory Practical

ERIC Educational Resources Information Center

Graham, Kate J.; Johnson, Brian J.; Jones, T. Nicholas; McIntee, Edward J.; Schaller, Chris P.

2008-01-01

An open-ended laboratory practical has been developed that challenges students to evaluate when different purification techniques are appropriate. In contrast to most lab practicals, the overall grade includes an evaluation of spectral analysis as well as writing skills. However, a significant portion of the grade lies in successful execution of a…

Microstructure Analyses of NA-Nanodiamond Particles

DTIC Science & Technology

2016-08-01

approximately 5 to 6 nm in diameter, similar to those obtained by distilled water purification . The energy dispersive analyzer from these perfectly well...NOTES 14. ABSTRACT The purification process of detonation diamond nanoparticles was perfectly accomplished using nitric acid at high...pressure nitric acid, whereas the previous detonation diamond nanoparticle was washed with distilled water and purified by oxidation

Purification and Characterization of Enzymes from Yeast: An Extended Undergraduate Laboratory Sequence for Large Classes

ERIC Educational Resources Information Center

Johanson, Kelly E.; Watt, Terry J.; McIntyre, Neil R.; Thompson, Marleesa

2013-01-01

Providing a project-based experience in an undergraduate biochemistry laboratory class can be complex with large class sizes and limited resources. We have designed a 6-week curriculum during which students purify and characterize the enzymes invertase and phosphatase from bakers yeast. Purification is performed in two stages via ethanol…

One-step purification of nisin A by immunoaffinity chromatography.

PubMed Central

Suárez, A M; Azcona, J I; Rodríguez, J M; Sanz, B; Hernández, P E

1997-01-01

The lantibiotic nisin A was purified to homogeneity by a single-step immunoaffinity chromatography method. An immunoadsorption matrix was developed by direct binding of anti-nisin A monoclonal antibodies to N-hydroxysuccinimide-activated Sepharose. The purification procedure was rapid and reproducible and rendered much higher final yields of nisin than any other described method. PMID:9406424

Quantitative evaluation of his-tag purification and immunoprecipitation of tristetraprolin and its mutant proteins from transfected human cells

USDA-ARS?s Scientific Manuscript database

Histidine (His)-tag is widely used for affinity purification of recombinant proteins, but the yield and purity of expressed proteins are quite different. Little information is available about quantitative evaluation of this procedure. The objective of the current study was to evaluate the His-tag pr...

Teaching Protein Purification and Characterization Techniques: A Student-Initiated, Project-Oriented Biochemistry Laboratory Course

ERIC Educational Resources Information Center

MacDonald, Gina

2008-01-01

This report describes a biochemistry laboratory that is completely project-oriented. Upper-level biology and chemistry majors work in teams to purify a protein of their choice. After the student groups have completed literature searches, ordered reagents, and made buffers they continue to learn basic protein purification and biochemical techniques…

Purification and Characterization of Taq Polymerase: A 9-Week Biochemistry Laboratory Project for Undergraduate Students

ERIC Educational Resources Information Center

Bellin, Robert M.; Bruno, Mary K.; Farrow, Melissa A.

2010-01-01

We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of "Thermus aquaticus" DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single…

SIPCAn (Separation, Isolation, Purification, Characterization, and Analysis): A One-Term, Integrated Project for the Undergraduate Organic Laboratory

ERIC Educational Resources Information Center

Dintzner, Matthew R.; Kinzie, Charles R.; Pulkrabek, Kimberly A.; Arena, Anthony F.

2011-01-01

SIPCAn, an acronym for separation, isolation, purification, characterization, and analysis, is presented as a one-term, integrated project for the first-term undergraduate organic laboratory course. Students are assigned two mixtures of unknown organic compounds--a mixture of two liquid compounds and a mixture of two solid compounds--at the…

Chemical purification of lanthanides for low-background experiments

NASA Astrophysics Data System (ADS)

Boiko, R. S.

2017-10-01

There are many potentially active isotopes among the lanthanide elements which are possible to use for low-background experiments to search for double β decay, dark matter, to investigate rare α and β decays. These kind of experiments require very low level of radioactive contamination, but commercially available compounds of lanthanides are always contamined by uranium, thorium, radium, potassium, etc. A simple chemical method based on liquid-liquid extraction has been applied for the purification of CeO2, Nd2O3 and Gd˙2O˙3 from radioactive traces. Detailed schemes of purification procedure are described. Measurements by using HPGe spectrometry demonstrate high efficiency in K, Ra, Th, U contaminations reduction on at least one order of magnitude.

[Study on extraction and purification process of total ginsenosides from Radix Ginseng].

PubMed

Xie, Li-Ling; Ren, Li; Lai, Xian-Sheng; Cao, Jun-Hui; Mo, Quan-Yi; Chen, Wei-Wen

2009-10-01

To optimize the technological parameters of the extraction and purification process of total ginsenosides from Radix Ginseng. With the contents of ginsenoside Rg1, ginsenoside Re and ginsenoside Rb1, the orthogonal design was adopted to optimize the extraction process. The purification process was studied by optimizing the elutive ratio of total ginsenosides as the marker. HPLC and spectrophotometer were employed for the study. The optimum conditions were as follows:Using 8 times volume of 75% ethanol extracting for 120 minutes and 2 times, the extraction temperature was 85 degrees C. AB-8 macroporous resin was selected, and the eluant was 4 BV 70% ethanol. The optimal conditions of extracting and purifying the total ginsenosides from Radix Ginseng is feasible.

[Studies on the extraction and purification of total saponins from Parched Semen Ziziphi Spinosae].

PubMed

Wu, Yulan; Ding, Anwei; Bao, Beihua

2005-03-01

To study the extraction and purification process of the total saponin from Parched Semen Ziziphi Spinosae with ethanol and macroporous resin. The total saponins were extracted with ethanol and purified with macroporous resin by orthogonal design, taking content and purity of jujuboside A as guideline. The optimum extraction condition was adding 6 times amount of 80% ethanol and refluxing 3 times, for 30 minutes each time. The purification process with macroporous resin HPD-100 was using 0.5% NaOH (150ml), 30% ethanol (150ml) to wash out impurity, and 70% ethanol 50 ml to wash out saponin. The purity of jujuboside A was up to 17.9% and the eluted ratio 72.8%.

Current submarine atmosphere control technology.

PubMed

Mazurek, W

1998-01-01

Air purification in submarines was introduced towards the end of World War II and was limited to the use of soda lime for the removal of carbon dioxide and oxygen candles for the regeneration of oxygen. The next major advances came with the advent of nuclear-powered submarines. These included the development of regenerative and, sometimes, energy-intensive processes for comprehensive atmosphere revitalization. With the present development of conventional submarines using air-independent propulsion there is a requirement for air purification similar to that of the nuclear-powered submarines but it is constrained by limited power and space. Some progress has been made in the development of new technology and the adoption of air purification equipment used in the nuclear-powered submarines for this application.

Application of visual basic in high-throughput mass spectrometry-directed purification of combinatorial libraries.

PubMed

Li, B; Chan, E C Y

2003-01-01

We present an approach to customize the sample submission process for high-throughput purification (HTP) of combinatorial parallel libraries using preparative liquid chromatography electrospray ionization mass spectrometry. In this study, Visual Basic and Visual Basic for Applications programs were developed using Microsoft Visual Basic 6 and Microsoft Excel 2000, respectively. These programs are subsequently applied for the seamless electronic submission and handling of data for HTP. Functions were incorporated into these programs where medicinal chemists can perform on-line verification of the purification status and on-line retrieval of postpurification data. The application of these user friendly and cost effective programs in our HTP technology has greatly increased our work efficiency by reducing paper work and manual manipulation of data.

Entanglement concentration and purification of two-mode squeezed microwave photons in circuit QED

NASA Astrophysics Data System (ADS)

Zhang, Hao; Alsaedi, Ahmed; Hayat, Tasawar; Deng, Fu-Guo

2018-04-01

We present a theoretical proposal for a physical implementation of entanglement concentration and purification protocols for two-mode squeezed microwave photons in circuit quantum electrodynamics (QED). First, we give the description of the cross-Kerr effect induced between two resonators in circuit QED. Then we use the cross-Kerr media to design the effective quantum nondemolition (QND) measurement on microwave-photon number. By using the QND measurement, the parties in quantum communication can accomplish the entanglement concentration and purification of nonlocal two-mode squeezed microwave photons. We discuss the feasibility of our schemes by giving the detailed parameters which can be realized with current experimental technology. Our work can improve some practical applications in continuous-variable microwave-based quantum information processing.

Study on a new water purification equipment with spiral lamellas

NASA Astrophysics Data System (ADS)

Feng, X. R.

2017-08-01

A new water purification equipment was introduced, especially the section of spiral lamellas. Utilization of spiral lamellas made the sedimentation space reach to 100%, not only improving sedimentation efficiency and reducing the cover space, but also saving investment. Production test results showed that the new water purification equipment with spiral lamellas had characteristics of excellent treatment efficiency and high shock resistant capacity. As the treatment water volume was 240 m3/d, when the turbidity, CODMn and UV254 were 203 NTU, 1.90 mg/L and 0.030 cm-1 in raw water, they were 0.32 NTU, 0.72mg/L and 0.011 cm-1 respectively in effluent water, which could fully meet the drinking water hygiene requirement.

Histamine monolith versatility to purify supercoiled plasmid deoxyribonucleic acid from Escherichia coli lysate.

PubMed

Sousa, A; Almeida, A M; Černigoj, U; Sousa, F; Queiroz, J A

2014-08-15

Preparation of high quantities of supercoiled plasmid DNA of pharmaceutical grade purity is a research area where intensive investigation is being performed. From this standpoint, several downstream methods have been proposed, among them the monolithic chromatographic strategies owing to excellent mass transfer properties of monolithic supports and their high binding capacity for large biomolecules. The present study explores the physicochemical properties of histamine ligand in a supercoiled plasmid DNA purification process from an Escherichia coli clarified lysate, where the emphasis is given to the elution strategy that allows higher selectivity and efficient removal of other impurities besides the open circular isoform. The combination of high NaCl concentration and acidic pH allowed the elimination of 89% of RNA during the preparative loading of the lysate sample. The results of the purification strategy with ascending sodium chloride gradient revealed that 97% of supercoiled plasmid DNA was recovered with a purity degree of 99%. In addition, using a combined purification strategy with ascending sodium chloride (capture step) and then descending ammonium sulfate (polishing step) gradient, it was achieved a lower supercoiled plasmid DNA recovery yield of 79% with a purity degree of 92%, although the dynamic binding capacity under these conditions was higher than in the previous strategy. A significant reduction of host contents, such as proteins, RNA and genomic DNA, was obtained in both purification strategies. Accordingly, histamine is a useful and versatile ligand that allows the desirable supercoiled plasmid purification with high yield and purity level. Copyright © 2014. Published by Elsevier B.V.

Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system.

PubMed

Barbosa, José Murillo P; Souza, Ranyere L; Fricks, Alini T; Zanin, Gisella Maria; Soares, Cleide Mara F; Lima, Alvaro S

2011-12-15

This work discusses the application of an aqueous two-phase system for the purification of lipases produced by Bacillus sp. ITP-001 using polyethylene glycol (PEG) and potassium phosphate. In the first step, the protein content was precipitated with ammonium sulphate (80% saturation). The enzyme remained in the aqueous solution and was dialyzed against ultra-pure water for 18 h and used to prepare an aqueous two-phase system (PEG/potassium phosphate). The use of different molecular weights of PEG to purify the lipase was investigated; the best purification factor (PF) was obtained using PEG 20,000g/mol, however PEG 8000 was used in the next tests due to lower viscosity. The influence of PEG and potassium phosphate concentrations on the enzyme purification was then studied: the highest FP was obtained with 20% of PEG and 18% of potassium phosphate. NaCl was added to increase the hydrophobicity between the phases, and also increased the purification factor. The pH value and temperature affected the enzyme partitioning, with the best purifying conditions achieved at pH 6.0 and 4°C. The molecular mass of the purified enzyme was determined to be approximately 54 kDa by SDS-PAGE. According to the results the best combination for purifying the enzyme is PEG 8000g/mol and potassium phosphate (20/18%) with 6% of NaCl at pH 6.0 and 4°C (201.53 fold). The partitioning process of lipase is governed by the entropy contribution. Copyright © 2011 Elsevier B.V. All rights reserved.

Secretory immunoglobulin purification from whey by chromatographic techniques.

PubMed

Matlschweiger, Alexander; Engelmaier, Hannah; Himmler, Gottfried; Hahn, Rainer

2017-08-15

Secretory immunoglobulins (SIg) are a major fraction of the mucosal immune system and represent potential drug candidates. So far, platform technologies for their purification do not exist. SIg from animal whey was used as a model to develop a simple, efficient and potentially generic chromatographic purification process. Several chromatographic stationary phases were tested. A combination of two anion-exchange steps resulted in the highest purity. The key step was the use of a small-porous anion exchanger operated in flow-through mode. Diffusion of SIg into the resin particles was significantly hindered, while the main impurities, IgG and serum albumin, were bound. In this step, initial purity was increased from 66% to 89% with a step yield of 88%. In a second anion-exchange step using giga-porous material, SIg was captured and purified by step or linear gradient elution to obtain fractions with purities >95%. For the step gradient elution step yield of highly pure SIg was 54%. Elution of SIgA and SIgM with a linear gradient resulted in a step yield of 56% and 35%, respectively. Overall yields for both anion exchange steps were 43% for the combination of flow-through and step elution mode. Combination of flow-through and linear gradient elution mode resulted in a yield of 44% for SIgA and 39% for SIgM. The proposed process allows the purification of biologically active SIg from animal whey in preparative scale. For future applications, the process can easily be adopted for purification of recombinant secretory immunoglobulin species. Copyright © 2017 Elsevier B.V. All rights reserved.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase.

PubMed

Trigoso, Yvonne D; Evans, Russell C; Karsten, William E; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5'and 3' terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40-50 mgs of protein, an improvement on the previous protein expression and multistep purification.

Cloning, Expression, and Purification of Histidine-Tagged Escherichia coli Dihydrodipicolinate Reductase

PubMed Central

Trigoso, Yvonne D.; Evans, Russell C.; Karsten, William E.; Chooback, Lilian

2016-01-01

The enzyme dihydrodipicolinate reductase (DHDPR) is a component of the lysine biosynthetic pathway in bacteria and higher plants. DHDPR catalyzes the NAD(P)H dependent reduction of 2,3-dihydrodipicolinate to the cyclic imine L-2,3,4,5,-tetrahydropicolinic acid. The dapB gene that encodes dihydrodipicolinate reductase has previously been cloned, but the expression of the enzyme is low and the purification is time consuming. Therefore the E. coli dapB gene was cloned into the pET16b vector to improve the protein expression and simplify the purification. The dapB gene sequence was utilized to design forward and reverse oligonucleotide primers that were used to PCR the gene from Escherichia coli genomic DNA. The primers were designed with NdeI or BamHI restriction sites on the 5’and 3’ terminus respectively. The PCR product was sequenced to confirm the identity of dapB. The gene was cloned into the expression vector pET16b through NdeI and BamHI restriction endonuclease sites. The resulting plasmid containing dapB was transformed into the bacterial strain BL21 (DE3). The transformed cells were utilized to grow and express the histidine-tagged reductase and the protein was purified using Ni-NTA affinity chromatography. SDS/PAGE gel analysis has shown that the protein was 95% pure and has approximate subunit molecular weight of 28 kDa. The protein purification is completed in one day and 3 liters of culture produced approximately 40–50 mgs of protein, an improvement on the previous protein expression and multistep purification. PMID:26815040

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.

Immunoaffinity based methods are superior to kits for purification of prostate derived extracellular vesicles from plasma samples.

PubMed

Brett, Sabine I; Lucien, Fabrice; Guo, Charles; Williams, Karla C; Kim, Yohan; Durfee, Paul N; Brinker, C J; Chin, Joseph I; Yang, Jun; Leong, Hon S

2017-05-01

The ability to isolate extracellular vesicles (EVs) such as exosomes or microparticles is an important method that is currently not standardized. While commercially available kits offer purification of EVs from biofluids, such purified EV samples will also contain non-EV entities such as soluble protein and nucleic acids that could confound subsequent experimentation. Ideally, only EVs would be isolated and no soluble protein would be present in the final EV preparation. We compared commercially available EV isolation kits with immunoaffinity purification techniques and evaluated our final EV preparations using atomic force microscopy (AFM) and nanoscale flow cytometry (NFC). AFM is the only modality capable of detecting distinguishing soluble protein from EVs which is important for downstream proteomics approaches. NFC is the only technique capable of quantitating the proportion of target EVs to non-target EVs in the final EV preparation. To determine enrichment of prostate derived EVs relative to non-target MPs, anti-PSMA (Prostate Specific Membrane Antigen) antibodies were used in NFC. Antibody-based immunoaffinity purification generated the highest quality of prostate derived EV preparations due to the lack of protein and RNA present in the samples. All kits produced poor purity EV preparations that failed to deplete the sample of plasma protein. While attractive due to their ease of use, EV purification kits do not provide substantial improvements in isolation of EVs from biofluids such as plasma. Immunoaffinity approaches are more efficient and economical and will also eliminate a significant portion of plasma proteins which is necessary for downstream approaches. © 2017 Wiley Periodicals, Inc.

Virtual Protein Purification: A Simple Exercise to Introduce pH as A Parameter That Effects Ion Exchange Chromatography

ERIC Educational Resources Information Center

Clark, Daniel D.; Edwards, Daniel J.

2018-01-01

This article describes a simple exercise using a free, easy-to-use, established online program. The exercise helps to reinforce protein purification concepts and introduces undergraduates to pH as a parameter that affects anion-exchange chromatography. The exercise was tested with biochemistry majors at California State University-Chico. Given the…

Radon assay and purification techniques

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simgen, Hardy

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.

Radon assay and purification techniques

NASA Astrophysics Data System (ADS)

Simgen, Hardy

2013-08-01

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 222Rn-daughters and the question of gas purification from radon is addressed.

Ligand-modified metal clusters for gas separation and purification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okrut, Alexander; Ouyang, Xiaoying; Runnebaum, Ron

2017-02-21

Provided is an organic ligand-bound metal surface that selects one gaseous species over another. The species can be closely sized molecular species having less than 1 Angstrom difference in kinetic diameter. In one embodiment, the species comprise carbon monoxide and ethylene. Such organic ligand-bound metal surfaces can be successfully used in gas phase separations or purifications, sensing, and in catalysis.

Purification of HgI.sub.2 for nuclear detector fabrication

DOEpatents

Schieber, Michael M.

1978-01-01

A process for purification of mercuric iodide (HgI.sub.2) to be used as a source material for the growth of detector quality crystals. The high purity HgI.sub.2 raw material is produced by a combination of three stages: synthesis of HgI.sub.2 from Hg and I.sub.2, repeated sublimation, and zone refining.

Improving Thai Students' Understanding of Concepts in Protein Purification by Using Thai and English Versions of a Simulation Program

ERIC Educational Resources Information Center

Phornphisutthimas, Somkiat; Panijpan, Bhinyo; Wood, Edward J.; Booth, Andrew G.

2007-01-01

To support student learning in biochemistry and related courses, a simulation program, the Protein Purification Program, offers an alternative multimedia-based tool. This program has now been translated to produce a Thai version. However, translation from the original into the Thai language is limited by the differences between the language…

Purification and Biochemical Characterization of Glutathione S-Transferase from Down Syndrome and Normal Children Erythrocytes: A Comparative Study

ERIC Educational Resources Information Center

Hamed, Ragaa R.; Maharem, Tahany M.; Abdel-Meguid, Nagwa; Sabry, Gilane M.; Abdalla, Abdel-Monem; Guneidy, Rasha A.

2011-01-01

Down syndrome (DS) is the phenotypic manifestation of trisomy 21. Our study was concerned with the characterization and purification of glutathione S-transferase enzyme (GST) from normal and Down syndrome (DS) erythrocytes to illustrate the difference in the role of this enzyme in the cell. Glutathione S-transferase and glutathione (GSH) was…

High-Throughput Kinetic Characterization of Ricin Toxin B Chain and Ovalbumin Antibodies Using Surface Plasmon Resonance

DTIC Science & Technology

2013-10-01

antibodies were purified using protein A purification, desalted in 1× phosphate buffered saline using Sephadex G-25 columns, and then filtered with a 0.2 µm...Purification of clones 2  The following materials were used in this process: o Protein A XK 16/15 (30 mL) column o Desalting Sephadex G

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.

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,…

A Novel Active-Learning Protein Purification Exercise for Large-Enrollment Introductory Biochemistry Courses Using the CHROM Web Applet

ERIC Educational Resources Information Center

Barrette-Ng, Isabelle H.; Usher, Ken C.

2013-01-01

The CHROM Web applet has been used to create a new active-learning exercise in which students design a purification scheme for a recombinant protein using ion-exchange chromatography (IEC). To successfully complete the exercise, students are challenged to apply elementary concepts from acid-base chemistry as well as protein and amino acid…

High-throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli

PubMed Central

Bruni, Renato

2014-01-01

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression and screening of membrane proteins using high throughput methodologies developed in our laboratory. Basic Protocol 1 deals with the cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that express at the miniscale, basic protocols 3 and 4 outline the methods employed for the expression and purification of targets at the midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. PMID:24510647

Using an FPLC to promote active learning of the principles of protein structure and purification.

PubMed

Robinson, Rebekah L; Neely, Amy E; Mojadedi, Wais; Threatt, Katie N; Davis, Nicole Y; Weiland, Mitch H

2017-01-02

The concepts of protein purification are often taught in undergraduate biology and biochemistry lectures and reinforced during laboratory exercises; however, very few reported activities allow students to directly gain experience using modern protein purification instruments, such as Fast Protein Liquid Chromatography (FPLC). This laboratory exercise uses size exclusion chromatography (SEC) and ion exchange (IEX) chromatography to separate a mixture of four different proteins. Students use an SEC chromatogram and corresponding SDS-PAGE gel to understand how protein conformations change under different conditions (i.e. native and non-native). Students explore strategies to separate co-eluting proteins by IEX chromatography. Using either cation or anion exchange, one protein is bound to the column while the other is collected in the flow-through. In this exercise, undergraduate students gain hands-on experience with experimental design, buffer and sample preparation, and implementation of instrumentation that is commonly used by experienced researchers while learning and applying the fundamental concepts of protein structure, protein purification, and SDS-PAGE. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(1):60-68, 2017. © 2016 The International Union of Biochemistry and Molecular Biology.

Selective Precipitation and Purification of Monovalent Proteins Using Oligovalent Ligands and Ammonium Sulfate

PubMed Central

Mirica, Katherine A.; Lockett, Matthew R.; Snyder, Phillip W.; Shapiro, Nathan D.; Mack, Eric T.; Nam, Sarah; Whitesides, George M.

2012-01-01

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: i) the removal of high-molecular weight impurities through the addition of ammonium sulfate to the crude cell lysate; ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins—for which appropriate oligovalent ligands can be synthesized—and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation. PMID:22188202

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate.

PubMed

Mirica, Katherine A; Lockett, Matthew R; Snyder, Phillip W; Shapiro, Nathan D; Mack, Eric T; Nam, Sarah; Whitesides, George M

2012-02-15

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.

Purification processes of xenogeneic bone substitutes and their impact on tissue reactions and regeneration.

PubMed

Perić Kačarević, Zeljka; Kavehei, Faraz; Houshmand, Alireza; Franke, Jörg; Smeets, Ralf; Rimashevskiy, Denis; Wenisch, Sabine; Schnettler, Reinhard; Jung, Ole; Barbeck, Mike

2018-04-01

Xenogeneic bone substitute materials are widely used in oral implantology. Prior to their clinical use, purification of the former bone tissue has to be conducted to ensure the removal of immunogenic components and pathogens. Different physicochemical methods are applied for purification of the donor tissue, and temperature treatment is one of these methods. Differences in these methods and especially the application of different temperatures for purification may lead to different material characteristics, which may influence the tissue reactions to these materials and the related (bone) healing process. However, little is known about the different material characteristics and their influences on the healing process. Thus, the aim of this mini-review is to summarize the preparation processes and the related material characteristics, safety aspects, tissue reactions, resorbability and preclinical and clinical data of two widely used xenogeneic bone substitutes that mainly differ in the temperature treatment: sintered (cerabone ® ) and non-sintered (Bio-Oss ® ) bovine-bone materials. Based on the summarized data from the literature, a connection between the material-induced tissue reactions and the consequences for the healing processes are presented with the aim of translation into their clinical application.

A Dual Protease Approach for Expression and Affinity Purification of Recombinant Proteins

PubMed Central

Raran-Kurussi, Sreejith; Waugh, David S.

2016-01-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification. PMID:27105777

Waste water purification using new porous ceramics prepared by recycling waste glass and bamboo charcoal

NASA Astrophysics Data System (ADS)

Nishida, Tetsuaki; Morimoto, Akane; Yamamoto, Yoshito; Kubuki, Shiro

2017-12-01

New porous ceramics (PC) prepared by recycling waste glass bottle of soft drinks (80 mass%) and bamboo charcoal (20 mass%) without any binder was applied to the waste water purification under aeration at 25 °C. Artificial waste water (15 L) containing 10 mL of milk was examined by combining 15 mL of activated sludge and 750 g of PC. Biochemical oxygen demand (BOD) showed a marked decrease from 178 to 4.0 (±0.1) mg L-1 in 5 days and to 2.0 (±0.1) mg L-1 in 7 days, which was equal to the Environmental Standard for the river water (class A) in Japan. Similarly, chemical oxygen demand (COD) decreased from 158 to 3.6 (±0.1) mg L-1 in 5 days and to 2.2 (±0.1) mg L-1 in 9 days, which was less than the Environmental Standard for the Seawater (class B) in Japan: 3.0 mg L-1. These results prove the high water purification ability of the PC, which will be effectively utilized for the purification of drinking water, fish preserve water, fish farm water, etc.

Optimization of alginate purification using polyvinylidene difluoride membrane filtration: Effects on immunogenicity and biocompatibility of three-dimensional alginate scaffolds

PubMed Central

Sondermeijer, Hugo P; Witkowski, Piotr; Woodland, David; Seki, Tetsunori; Aangenendt, Frank J; van der Laarse, Arnoud; Itescu, Silviu; Hardy, Mark A

2017-01-01

Sodium alginate is an effective biomaterial for tissue engineering applications. Non-purified alginate is contaminated with protein, lipopolysaccharide, DNA, and RNA, which could elicit adverse immunological reactions. We developed a purification protocol to generate biocompatible alginate based on (a) activated charcoal treatment, (b) use of hydrophobic membrane filtration (we used hydrophobic polyvinylidene difluoride membranes to remove organic contaminants), (c) dialysis, and finally (d) ethanol precipitation. Using this approach, we could omit pre-treatment with chloroform and significantly reduce the quantities of reagents used. Purification resulted in reduction of residual protein by 70% down to 0.315 mg/g, DNA by 62% down to 1.28 μg/g, and RNA by 61% down to less than 10 μg/g, respectively. Lipopolysaccharide levels were reduced by >90% to less than 125 EU/g. Purified alginate did not induce splenocyte proliferation in vitro. Three-dimensional scaffolds generated from purified alginate did not elicit a significant foreign body reaction, fibrotic overgrowth, or macrophage infiltration 4 weeks after implantation. This study describes a simplified and economical alginate purification method that results in alginate purity, which meets clinically useful criteria. PMID:27114440

Preparative Purification of Polyphenols from Aronia melanocarpa (Chokeberry) with Cellular Antioxidant and Antiproliferative Activity.

PubMed

Gao, Ningxuan; Wang, Yuehua; Jiao, Xinyao; Chou, Shurui; Li, Enhui; Li, Bin

2018-01-10

The aim of this study was the purification process of polyphenols from Aronia melanocarpa (chokeberry), and the purification parameters were optimised by adsorption and desorption tests. By comparing adsorption and desorption ability of polyphenols from chokeberry on six kinds of macroporous resin, XAD-7 resin was selected. Experiments prove that the best purification parameters of static adsorption and desorption were sample pH = 4.0 with 4 h of adsorption; and desorption solvent is 95% ethanol (pH = 7.0) with 2 h of desorption. The best dynamic parameters were 9.3 bed volume (BV) of sample loading amount at a feeding flow rate of 2 BV/h, and washing the column with 5.8 BV of water, followed by subsequent elution with an eluent volume of 5.0 mL at an elution flow rate of 2 BV/h. Next the antioxidant and antiproliferative activity of polyphenols from chokeberry, blueberries, haskap berries was studied on HepG2 human liver cancer cells. The results show that polyphenol from chokeberry has a strong antioxidant effect. Taking into account the content of polyphenols in fruit, polyphenols from chokeberry represent a very valuable natural antioxidant source with antiproliferative products.

Effects of indoor air purification by an air cleaning system (Koala technology) on semen parameters in male factor infertility: results of a pilot study.

PubMed

Paradisi, R; Vanella, S; Barzanti, R; Cani, C; Battaglia, C; Seracchioli, R; Venturoli, S

2009-06-01

A number of studies indicated a clear decline in semen quality in the past 30-50 years and there is accumulating evidence that this decline might result from exposure to high levels of air pollution. To examine the impact of environment on male reproductive ability, we undertook for the first time a pilot study on semen quality of infertile men exposed to purification of indoor air. Ten subjects with a history of unexplained male infertility and poor semen quality were exposed for at least 1 year to a cleaning indoor air system (Koala technology). The key feature of this air purifier is the unique innovative multiple filtering system. The treatment of total purification of indoor air showed neither improvements in semen parameters nor variation in reproductive hormones (P = N.S.), but induced an evident increase (P < 0.03 and more) in seminal leucocytic concentrations. Within the limits due to the small sample of subjects recruited, the sole purification of indoor air does not seem enough to improve semen quality, although the increase in leucocytic concentrations could indicate an activation of the role of immunosurveillance in a purified indoor air environment.

Research on the Purification Effect of Aquatic Plants Based on Grey Clustering Method

NASA Astrophysics Data System (ADS)

Gu, Sudan; Du, Fuhui

2018-01-01

This paper uses the grey clustering method to evaluate the water quality level of the MingGuan constructed wetland at the import and export of artificial wetlands. Constructed wetland of Ming Guanis established on the basis of the Fuyang River’s water quality improvement, to choose suitable aquatic plants, in order to achieve the Fuyang River water purification effect. Namely TP, TN, NH3-N, DO, COD and COMMn and permanganate index are selected as clustering indicators. Water quality is divided into five grades according to the Surface Water Environmental Quality Standard (GB3838-2002) as the evaluation standard. In order to select the suitable wetland plants, the purification effect of 6 kinds of higher aquatic plants on the sewage of fuyang river was tested. one kind of plants was selected: Typha. The results show that the water quality of the section is gradually changed from V water quality to III water quality. After tartificial wetland of cycle for a long time, Typha has good purification effect. In November, water quality categories are basically concentrated in the VI, V class, may be caused by chemical decomposition of aquatic plants, should strengthen the academic research.

Evaluation of immobilized metal-ion affinity chromatography (IMAC) as a technique for IgG(1) monoclonal antibodies purification: the effect of chelating ligand and support.

PubMed

Bresolin, I T L; Borsoi-Ribeiro, M; Tamashiro, W M S C; Augusto, E F P; Vijayalakshmi, M A; Bueno, S M A

2010-04-01

Monoclonal antibodies (MAbs) have been used for therapies and some analytical procedures as highly purified molecules. Many techniques have been applied and studied, focusing on monoclonal antibodies purification. In this study, an immobilized metal affinity chromatography membrane was developed and evaluated for the purification of anti-TNP IgG(1) mouse MAbs from cell culture supernatant after precipitation with a 50% saturated ammonium sulfate solution. The chelating ligands iminodiacetic acid, carboxymethylated aspartic acid (CM-Asp), nitrilotriacetic acid, and tris (carboxymethyl) ethylenediamine in agarose gels with immobilized Ni(II) and Zn(II) ions were compared for the adsorption and desorption of MAbs. The most promising chelating ligand--CM-Asp--was then coupled to poly(ethylene vinyl alcohol) (PEVA) hollow fiber membranes. According to SDS-PAGE and ELISA analyses, a higher selectivity and a purification factor of 85.9 (fraction eluted at 500 mM Tris) were obtained for IgG(1) using PEVA-CM-Asp-Zn(II). The anti-TNP MAb could be eluted under mild pH conditions causing no loss of antigen binding capacity.

Two novel solvent system compositions for protected synthetic peptide purification by centrifugal partition chromatography.

PubMed

Amarouche, Nassima; Giraud, Matthieu; Forni, Luciano; Butte, Alessandro; Edwards, F; Borie, Nicolas; Renault, Jean-Hugues

2014-04-11

Protected synthetic peptide intermediates are often hydrophobic and not soluble in most common solvents. They are thus difficult to purify by preparative reversed-phase high-performance liquid chromatography (RP-HPLC), usually used for industrial production. It is then challenging to develop alternative chromatographic purification processes. Support-free liquid-liquid chromatographic techniques, including both hydrostatic (centrifugal partition chromatography or CPC) and hydrodynamic (counter-current chromatography or CCC) devices, are mainly involved in phytochemical studies but have also been applied to synthetic peptide purification. In this framework, two new biphasic solvent system compositions covering a wide range of polarity were developed to overcome solubility problems mentioned above. The new systems composed of heptane/tetrahydrofuran/acetonitrile/dimethylsulfoxide/water and heptane/methyl-tetrahydrofuran/N-methylpyrrolidone/water were efficiently used for the CPC purification of a 39-mer protected exenatide (Byetta®) and a 8-mer protected peptide intermediate of bivalirudin (Angiox®) synthesis. Phase compositions of the different biphasic solvent systems were determined by (1)H nuclear magnetic resonance. Physico-chemical properties including viscosity, density and interfacial tension of these biphasic systems are also described. Copyright © 2014 Elsevier B.V. All rights reserved.

A dual protease approach for expression and affinity purification of recombinant proteins.

PubMed

Raran-Kurussi, Sreejith; Waugh, David S

2016-07-01

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

The modified swirl sedimentation tanks for water purification.

PubMed

Ochowiak, Marek; Matuszak, Magdalena; Włodarczak, Sylwia; Ancukiewicz, Małgorzata; Krupińska, Andżelika

2017-03-15

This paper discusses design, evaluation, and application for the use of swirl/vortex technologies as liquid purification system. A study was performed using modified swirl sedimentation tanks. The vortex separators (OW, OWK, OWR and OWKR) have been studied under laboratory conditions at liquid flow rate from 2.8⋅10 -5 to 5.1⋅10 -4 [m 3 /s]. The pressure drop and the efficiency of purification of liquid stream were analyzed. The suspended particles of different diameters were successfully removed from liquid with the application of swirl chambers of proposed constructions. It was found that damming of liquid in the tank increases alongside liquid stream at the inlet and depends on the tank construction. The efficiency of the sedimentation tanks increases alongside the diameters of solid particles and decrease in the liquid flow rate. The best construction proved to be the OWR sedimentation tank due to smallest liquid damming, even at high flow rates, and the highest efficiency of the purification liquid stream for solid particles of the smallest diameter. The proposed solution is an alternative to the classical constructions of sedimentation tanks. Copyright © 2016 Elsevier Ltd. All rights reserved.

Californium purification and electrodeposition

DOE PAGES

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

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 HNO 3 and HCl, and Eichrom TEVA resin in NH 4SCN. The TEVA NH 4SCN system was found to completely separate 145Sm and 153Gd from 249Cf and was adopted into the purification process used in purifying the 252Cf.« less

The Analysis of the System of special water purification of Beloyarskaya Nuclear Power Plant unit BN-800

NASA Astrophysics Data System (ADS)

Valtseva, A. I.; Bibik, I. S.

2017-11-01

This article discusses how the latest system of special water purification KPF-30, designed specifically for the fourth power unit of Beloyarskaya nuclear power plant, which has a number of advantages over other water purification systems as chemical-physical and technical-economic, environmental, and other industrial indicators. The scheme covered in this article systems of special water purification involves the use of a hydrocyclone at the preliminary stage of water treatment, as a worthy alternative to ion-exchange filters, which can significantly reduce the volume of toxic waste. The world community implements the project of closing the nuclear fuel cycle, there is a need to improve the reliability of the equipment for safe processes and development of critical and supercritical parameters in the nuclear industry. Essentially, on operated NPP units, the only factor that can cost-effectively optimize to improve the reliability of equipment is the water chemistry. System KPF30 meets the principles and criteria of ecological safety, demonstrating the justification for reagent less method of water treatment on the main stages, in which no formation of toxic wastes, leading to irreversible consequences of environmental pollution and helps to conserve water.

Expression, stabilization and purification of membrane proteins via diverse protein synthesis systems and detergents involving cell-free associated with self-assembly peptide surfactants.

PubMed

Zheng, Xuan; Dong, Shuangshuang; Zheng, Jie; Li, Duanhua; Li, Feng; Luo, Zhongli

2014-01-01

G-protein coupled receptors (GPCRs) are involved in regulating most of physiological actions and metabolism in the bodies, which have become most frequently addressed therapeutic targets for various disorders and diseases. Purified GPCR-based drug discoveries have become routine that approaches to structural study, novel biophysical and biochemical function analyses. However, several bottlenecks that GPCR-directed drugs need to conquer the problems including overexpression, solubilization, and purification as well as stabilization. The breakthroughs are to obtain efficient protein yield and stabilize their functional conformation which are both urgently requiring of effective protein synthesis system methods and optimal surfactants. Cell-free protein synthesis system is superior to the high yields and post-translation modifications, and early signs of self-assembly peptide detergents also emerged to superiority in purification of membrane proteins. We herein focus several predominant protein synthesis systems and surfactants involving the novel peptide detergents, and uncover the advantages of cell-free protein synthesis system with self-assembling peptide detergents in purification of functional GPCRs. This review is useful to further study in membrane proteins as well as the new drug exploration. Copyright © 2014 Elsevier Inc. All rights reserved.

Electron-stimulated purification of platinum nanostructures grown via focused electron beam induced deposition.

PubMed

Lewis, Brett B; Stanford, Michael G; Fowlkes, Jason D; Lester, Kevin; Plank, Harald; Rack, Philip D

2015-01-01

Platinum-carbon nanostructures deposited via electron beam induced deposition from MeCpPt(IV)Me3 are purified during a post-deposition electron exposure treatment in a localized oxygen ambient at room temperature. Time-dependent studies demonstrate that the process occurs from the top-down. Electron beam energy and current studies demonstrate that the process is controlled by a confluence of the electron energy loss and oxygen concentration. Furthermore, the experimental results are modeled as a 2nd order reaction which is dependent on both the electron energy loss density and the oxygen concentration. In addition to purification, the post-deposition electron stimulated oxygen purification process enhances the resolution of the EBID process due to the isotropic carbon removal from the as-deposited materials which produces high-fidelity shape retention.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pocar, Andrea, E-mail: pocar@umass.edu; Physics Division, Lawrence Livermore National Laboratory, Livermore, California 94550

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 aremore » addressed with particular focus on the experience gained with the EXO-200 neutrino-less double beta decay detector.« less

[Extraction, isolation and purification for ginkgolide B].

PubMed

Zhang, Chenfeng; Li, Minghui; Tang, Yun; Zhang, Yanhai; Shi, Min; Sheng, Longsheng

2010-08-01

To establish a simple extraction, isolation and purification method for ginkgolide B from ginkgo leaf. The optimum conditions of extraction, isolation and purification were studied by taking the transfer rate of ginkgolide B as index. Ginkgo leaf was extracted with 70% ethanol for three times, the extracts were concentrated to remove ethanol and diluted by water till the crude drug density reached 0.1 g x mL(-1). The dilution was adsorbed with HPD-450 macroporous resin. The impurities were eluted with 20% ethanol and ginkgolide B was eluted with 80% ethanol. Then the 80% ethanol eluant was concentrated and crystallized. Finally the crude crystals were recrystallized with isopropanol. The purity of the ginkgolide B recrystallization was 95%. The process was stable and easy to operate, which was suited to industrialized production.

Bioactivities, isolation and purification methods of polysaccharides from natural products: A review.

PubMed

Shi, Lei

2016-11-01

Polysaccharides play multiple roles and have extensive bioactivities in life process and an immense potential in healthcare, food and cosmetic industries, due to their therapeutic effects and relatively low toxicity. This review describes their major functions involved in antitumor, anti-virus, and anti-inflammatory bioactivities. Due to their enormous structural heterogeneity, the approaches for isolation and purification of polysaccharides are distinct from that of the other macromolecules such as proteins, etc. Yet, to achieve the homogeneity is the initial step for studies of polysaccharide structure, pharmacology, and its structure-activity relationships. According to the experiences accumulated by our lab and the published literatures, this review also introduces the methods widely used in isolation and purification of polysaccharides. Copyright © 2016 Elsevier B.V. All rights reserved.

Results from KamLAND-Zen

DOE PAGES

Asakura, K.; Gando, A.; Gando, Y.; ...

2015-07-15

KamLAND-Zen reports on a preliminary search for neutrinoless double-beta decay with 136Xe based on 114.8 live-days after the purification of the xenon loaded liquid scintillator. In this data, the problematic 110mAg background peak identified in previous searches is reduced by more than a factor of 10. By combining the KamLAND-Zen pre- and post-purification data, we obtain a preliminary lower limit on the 0νββ decay half-life of T 0ν 1/2 > 2.6×10 25 yr at 90% C.L. The search sensitivity will be enhanced with additional low background data after the purification. As a result, prospects for further improvements with future KamLAND-Zenmore » upgrades are also presented.« less

Development of a passive micromixer based on repeated fluid twisting and flattening, and its application to DNA purification.

PubMed

Lee, Nae Yoon; Yamada, Masumi; Seki, Minoru

2005-11-01

We have developed a three-dimensional passive micromixer based on new mixing principles, fluid twisting and flattening. This micromixer is constructed by repeating two microchannel segments, a "main channel" and a "flattened channel", which are very different in size and are arranged perpendicularly. At the intersection of these segments the fluid inside the micromixer is twisted and then, in the flattened channel, the diffusion length is greatly reduced, achieving high mixing efficiency. Several types of micromixer were fabricated and the effect of microchannel geometry on mixing performance was evaluated. We also integrated this micromixer with a miniaturized DNA purification device, in which the concentration of the buffer solution could be rapidly changed, to perform DNA purification based on solid-phase extraction.

Copper(I)/TEMPO Catalyzed Aerobic Oxidation of Primary Alcohols to Aldehydes with Ambient Air

PubMed Central

Hoover, Jessica M.; Steves, Janelle E.; Stahl, Shannon S.

2012-01-01

This protocol describes a practical laboratory-scale method for aerobic oxidation of primary alcohols to aldehydes, using a chemoselective CuI/TEMPO catalyst system. The catalyst is prepared in situ from commercially available reagents, and the reactions are performed in a common organic solvent (acetonitrile) with ambient air as the oxidant. Three different reaction conditions and three procedures for the isolation and purification of the aldehyde product are presented. The oxidations of eight different alcohols, described here, include representative examples of each reaction condition and purification method. Reaction times vary from 20 min to 24 h, depending on the alcohol, while the purification methods each take about 2 h. The total time necessary for the complete protocol ranges from 3 – 26 h. PMID:22635108

Hamiltonian purification

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orsucci, Davide; Burgarth, Daniel; Facchi, Paolo

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 purificationmore » and generator purification of an algebra are also introduced and optimal solutions for u(d) are provided.« less

Recent Methods for Purification and Structure Determination of Oligonucleotides.

PubMed

Zhang, Qiulong; Lv, Huanhuan; Wang, Lili; Chen, Man; Li, Fangfei; Liang, Chao; Yu, Yuanyuan; Jiang, Feng; Lu, Aiping; Zhang, Ge

2016-12-18

Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers.

Comparison of Influenza Virus Particle Purification Using Magnetic Sulfated Cellulose Particles with an Established Centrifugation Method for Analytics.

PubMed

Serve, Anja; Pieler, Michael Martin; Benndorf, Dirk; Rapp, Erdmann; Wolff, Michael Werner; Reichl, Udo

2015-11-03

A method for the purification of influenza virus particles using novel magnetic sulfated cellulose particles is presented and compared to an established centrifugation method for analytics. Therefore, purified influenza A virus particles from adherent and suspension MDCK host cell lines were characterized on the protein level with mass spectrometry to compare the viral and residual host cell proteins. Both methods allowed one to identify all 10 influenza A virus proteins, including low-abundance proteins like the matrix protein 2 and nonstructural protein 1, with a similar impurity level of host cell proteins. Compared to the centrifugation method, use of the novel magnetic sulfated cellulose particles reduced the influenza A virus particle purification time from 3.5 h to 30 min before mass spectrometry analysis.

Development of Purification Protocol Specific for Bacteriocin 105B

DTIC Science & Technology

2017-02-09

determined to exhibit activity against a pathogenic organism of interest to the Army, Bacillus anthracis Sterne, a surrogate of the active form of...employment in future assays and development into a platform, such as a textile, that relays antimicrobial activity . 15. SUBJECT TERMS 105B...Purification with Ion Exchange Column Chromatography. Representative activity drop test assay evaluating activity of fractions collected from ion

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. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Production of capsular polysaccharide of Streptococcus pneumoniae type 14 and its purification by affinity chromatography.

PubMed

Suárez, N; Fraguas, L F; Texeira, E; Massaldi, H; Batista-Viera, F; Ferreira, F

2001-02-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents.

Production of Capsular Polysaccharide of Streptococcus pneumoniae Type 14 and Its Purification by Affinity Chromatography

PubMed Central

Suárez, Norma; Fraguas, Laura Franco; Texeira, Esther; Massaldi, Hugo; Batista-Viera, Francisco; Ferreira, Fernando

2001-01-01

We describe a rapid and efficient method for producing the capsular polysaccharide of Streptococcus pneumoniae by fermentation on tryptic soy broth and purification of this compound by using immobilized soybean lectin as an affinity adsorbent. In principle, the same strategy can be used to produce purified capsular polysaccharides from other streptococcal serotypes by selecting the appropriate lectin adsorbents. PMID:11157270

Evaluation of Low-Pressure Drop Antimicrobial and Hybrid Air Filters

DTIC Science & Technology

2006-09-01

purification of aerosol- contaminated air streams has been performed by mechanical filtration. Existing particle filters will stop bacterial and viral...or hybrid low-∆P antimicrobial particulate filter materials. 1.2 Background Traditional purification of aerosol- contaminated air streams has...Plastics, Lima , Ohio). Each path runs through a test article and thence through one AGI-30 all-glass impinger (Chemglass, Vineland, N.J.) partially

Legionella pneumophila Toxin, Isolation and Purification

DTIC Science & Technology

1981-01-01

which dis- plays an in vivo lethality. The purification procedures involve acid precipitation, gel chromatography, and preparative isotachophoresis. The...Chymotrypsinogen A, Ribonuclease A, and Apoprotinin as markers. Preparation of antiserum One milliqram amounts of protein from Le jonella acid ...RESULTS Toxin isolation Step 1: Acid precipitation of crude toxin. 1.0 N HCl acid was slowly added to rapidly stirred crude toxin until pH 3.5 was

Purification of metal-organic framework materials

DOEpatents

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.

Chemical Protection Testing of Sorbent-Based Air Purification Components (APCs)

DTIC Science & Technology

2016-06-24

APC’s ability to filter air in a chemically contaminated environment. 15. SUBJECT TERMS Air purification component; APC; filtration fabric...FF, filter media, collective protection; individual protection. 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT SAR 18...incoming air. The intent of this process is to produce traceable, quantifiable, and defensible data that can be used to analyze an APC’s ability to filter

A Real-Time and Hands-On Research Course in Protein Purification and Characterization: Purification and Crystal Growth of Human Inosine Triphosphate Pyrophosphatase

ERIC Educational Resources Information Center

Kreiling, Jodi L.; Brader, Kerry; Kolar, Carol; Borgstahl, Gloria E. O.

2011-01-01

A new lecture/laboratory course to offer advanced biochemical training for undergraduate and early graduate students has been developed in the Department of Chemistry at the University of Nebraska at Omaha. This unique course offers students an opportunity to work hands-on with modern instrumentation not normally found in a predominately…

Purification of metal-organic framework materials

DOEpatents

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.

Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation and Air Purification

DTIC Science & Technology

2012-06-01

Eliasson, and M. Hirth, “ Ozone Generation from Oxygen and Air: Discharge Physics and Reaction Mechanisms,” Ozone Sci. and Eng., vol. 10, pp. 367-378...Phase I Final Report: Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation and Air Purification...Contract Number: FA9550-11-C-0087 June 2012 Low Cost, Efficient Microcavity Plasma Ozone Generation for Water Remediation

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…

Potential of using plant extracts for purification of shallow well water in Malawi

NASA Astrophysics Data System (ADS)

Pritchard, M.; Mkandawire, T.; Edmondson, A.; O'Neill, J. G.; Kululanga, G.

There has been very little scientific research work into the use of plant extracts to purify groundwater. Research studies on the purification of groundwater have mainly been carried out in developed countries and have focused on water purification systems using aluminium sulphate (a coagulant) and chlorine (a disinfectant). Such systems are expensive and not viable for rural communities due to abject poverty. Shallow well water, which is commonly available throughout Africa, is often grossly contaminated and usually consumed untreated. As a result, water-related diseases kill more than 5 million people every year worldwide. This research was aimed at examining natural plant extracts in order to develop inexpensive ways for rural communities to purify their groundwater. The study involved creating an inventory of plant extracts that have been used for water and wastewater purification. A prioritisation system was derived to select the most suitable extracts, which took into account criteria such as availability, purification potential, yield and cost of extraction. Laboratory trials were undertaken on the most promising plant extracts, namely: Moringa oleifera, Jatropha curcas and Guar gum. The extracts were added to water samples obtained from five shallow wells in Malawi. The trials consisted of jar tests to assess the coagulation potential and the resulting effect on physico-chemical and microbiological parameters such as temperature, pH, turbidity and coliforms. The results showed that the addition of M. oleifera, J. curcas and Guar gum can considerably improve the quality of shallow well water. Turbidity reduction was higher for more turbid water. A reduction efficiency exceeding 90% was achieved by all three extracts on shallow well water that had a turbidity of 49 NTU. A reduction in coliforms was about 80% for all extracts. The pH of the water samples increased with dosage, but remained within acceptable levels for drinking water for all the extracts. Overall, M. oleifera powder produced superior results, followed by Guar gum and lastly J. curcas. There is a need to carry out further more detailed tests, which include toxicity to guarantee the safety of using plant extracts as a coagulant in the purification of drinking water for human consumption.

Doing that thing that scientists do: A discovery-driven module on protein purification and characterization for the undergraduate biochemistry laboratory classroom.

PubMed

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, students develop research skills through work on an original research project and gain confidence in their ability to design and execute an experiment while faculty can enhance their scholarly pursuits through the acquisition of original data in the classroom laboratory. Students are prepared for a 6-8 week discovery-driven project on the purification of the Escherichia coli cytidylate kinase (CMP kinase) through in class problems and other laboratory exercises on bioinformatics and protein structure analysis. After a minimal amount of guidance on how to perform the CMP kinase in vitro enzyme assay, SDS-PAGE, and the basics of protein purification, students, working in groups of three to four, develop a protein purification protocol based on the scientific literature and investigate some aspect of CMP kinase that interests them. Through this process, students learn how to implement a new but perhaps previously worked out procedure to answer their research question. In addition, they learn the importance of keeping a clear and thorough laboratory notebook and how to interpret their data and use that data to inform the next set of experiments. Following this module, students had increased confidence in their ability to do basic biochemistry techniques and reported that the "self-directed" nature of this lab increased their engagement in the project. © 2015 The International Union of Biochemistry and Molecular Biology.

The purification step is not crucial in EIA measurements of thromboxane B2 and 11-dehydrothromboxane B2 in human plasma.

PubMed

Sadilkova, Lenka; Paluch, Zoltan; Mottlova, Jirina; Bednar, Frantisek; Alusik, Stefan

2012-01-01

Thromboxane B2 (TxB2) and particularly 11-dehydrothromboxane B2 (11-dTxB2) are widely used as prognostic risk markers of platelet activation in cardiovascular diseases. The main errors in TxB2 and 11-dTxB2 determination include either low concentrations of circulating TxB2 (1 - 2 pg/mL) and 11-dTxB2 (0.9 - 4.3 pg/mL) or rather high transiency (mean TxB2 half-life is approximately 5 minutes) as well as an incorrect pre-analytical phase set up. The aim of this study was to investigate the impact of a widely used purification step on the results of enzyme immunosorbent assay (EIA)--based measurement of the two selected thromboxanes. For the purpose of this study, 20 plasma samples (10 healthy donors, 10 patients under treatment with acetylsalicylic acid) were screened for TxB2 and 11-dTxB2 concentrations using commercial competitive EIA kits (Cayman Chemicals, Tallinn, Estonia; Neogen, Lexington, KY, USA) with or without the introduction of the purification procedure. The purification step does not significantly affect the results of EIA measurements of the two of TxA2 metabolites (TxB2, 11-dTxB2) in human plasma. The levels of TxB2 and 11-dTxB2 determined in the plasma samples were not significantly changed (p < 0.05) when the purification step was omitted compared to the purified samples. This study establishes a protocol allowing for reliable and reproducible plasma TxB2 and 11-dTxB2 EIA measurement for routine basic screening of platelet function.

Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

PubMed Central

Hey, Y; Dean, P D

1983-01-01

1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B. Images Fig. 1. PMID:6847623

Tandem dye-ligand chromatography and biospecific elution applied to the purification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides.

PubMed

Hey, Y; Dean, P D

1983-02-01

1. A total of 65 immobilized triazine dyes were screened for their ability to purify the dual-nucleotide-specific glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides. From this screen a 'negative' (Matrex Gel Purple A) and a 'positive' (Matrex Gel Orange B) adsorbent were found to be the best in terms of overall purification and yield and were therefore combined to give the best purification. 2. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides was purified approx. 56-fold in a two-step tandem chromatographic system using Matrex Gel Purple A followed by Matrex Gel Orange B chromatography to a specific activity of 228 units/mg of protein in a final yield of 73%. 3. A study of the elution characteristics of glucose-6-phosphate dehydrogenase bound to Matrex Gel Orange B by KCl (pulse and gradient) and biospecific eluents (pulse) was carried out. NADP+, NADPH and adenosine 2',5'-bisphosphate were found to be the only effective biospecific eluents. A pulse of 50 microM-NADP+ (1/2 column vol.) was found to give a better purification than a 0-1 M-KCl gradient and therefore was the preferred method of elution. 4. Presaturation of the enzyme with various nucleotides was carried out to determine the effect on the subsequent binding of glucose-6-phosphate dehydrogenase to Matrex Gel Orange B. The results of these and biospecific-elution studies lead us to propose two possible schemes to explain the mechanism of the dye-protein interaction. 5. Reusability, capacity of the adsorbent and effect of varying the ligand concentration were also studied in the purification of glucose-6-phosphate dehydrogenase on Matrex Gel Orange B.

Comparing postdeposition reactions of electrons and radicals with Pt nanostructures created by focused electron beam induced deposition.

PubMed

Spencer, Julie A; Barclay, Michael; Gallagher, Miranda J; Winkler, Robert; Unlu, Ilyas; Wu, Yung-Chien; Plank, Harald; McElwee-White, Lisa; Fairbrother, D Howard

2017-01-01

The ability of electrons and atomic hydrogen (AH) to remove residual chlorine from PtCl 2 deposits created from cis -Pt(CO) 2 Cl 2 by focused electron beam induced deposition (FEBID) is evaluated. Auger electron spectroscopy (AES) and energy-dispersive X-ray spectroscopy (EDS) measurements as well as thermodynamics calculations support the idea that electrons can remove chlorine from PtCl 2 structures via an electron-stimulated desorption (ESD) process. It was found that the effectiveness of electrons to purify deposits greater than a few nanometers in height is compromised by the limited escape depth of the chloride ions generated in the purification step. In contrast, chlorine atoms can be efficiently and completely removed from PtCl 2 deposits using AH, regardless of the thickness of the deposit. Although AH was found to be extremely effective at chemically purifying PtCl 2 deposits, its viability as a FEBID purification strategy is compromised by the mobility of transient Pt-H species formed during the purification process. Scanning electron microscopy data show that this results in the formation of porous structures and can even cause the deposit to lose structural integrity. However, this phenomenon suggests that the use of AH may be a useful strategy to create high surface area Pt catalysts and may reverse the effects of sintering. In marked contrast to the effect observed with AH, densification of the structure was observed during the postdeposition purification of PtC x deposits created from MeCpPtMe 3 using atomic oxygen (AO), although the limited penetration depth of AO restricts its effectiveness as a purification strategy to relatively small nanostructures.

The chromatography-free release, isolation and purification of recombinant peptide for fibril self-assembly.

PubMed

Hartmann, B M; Kaar, W; Yoo, I K; Lua, L H L; Falconer, R J; Middelberg, A P J

2009-12-01

One of the major expenses associated with recombinant peptide production is the use of chromatography in the isolation and purification stages of a bioprocess. Here we report a chromatography-free isolation and purification process for recombinant peptide expressed in Escherichia coli (E. coli). Initial peptide release is by homogenization and then by enzymatic cleavage of the peptide-containing fusion protein, directly in the E. coli homogenate. Release is followed by selective solvent precipitation (SSP) to isolate and purify the peptide away from larger cell contaminants. Specifically, we expressed in E. coli the self-assembling beta-sheet forming peptide P(11)-2 in fusion to thioredoxin. Homogenate was heat treated (55 degrees C, 15 min) and then incubated with tobacco etch virus protease (TEVp) to release P(11)-2 having a native N-terminus. SSP with ethanol at room temperature then removed contaminating proteins in an integrated isolation-purification step; it proved necessary to add 250 mM NaCl to homogenate to prevent P(11)-2 from partitioning to the precipitate. This process structure gave recombinant P(11)-2 peptide at 97% polypeptide purity and 40% overall yield, without a single chromatography step. Following buffer-exchange of the 97% pure product by bind-elute chromatography into defined chemical conditions, the resulting peptide was shown to be functionally active and able to form self-assembled fibrils. To the best of our knowledge, this manuscript reports the first published process for chromatography-free recombinant peptide release, isolation and purification. The process proved able to deliver functional recombinant peptide at high purity and potentially low cost, opening cost-sensitive materials applications for peptide-based materials.

Elastin-like polypeptides: A strategic fusion partner for biologics.

PubMed

Yeboah, Agnes; Cohen, Rick I; Rabolli, Charles; Yarmush, Martin L; Berthiaume, Francois

2016-08-01

Elastin-like peptides (ELPs) are derivatives of tropoelastin with a unique property that allows them to stay soluble below a certain critical temperature but reversibly form aggregates above that temperature. Since they are derived from tropoelastin, ELPs are biocompatible, non-toxic, and non-immunogenic. The unique properties of ELPs have made them a desirable class of fusion tags used in several biomedical applications including targeted drug delivery and enhancing the half-life of protein drugs. The most significant property of an ELP is that when fused to other proteins, the phase transition property of the ELP is maintained, and the target protein can be purified using the thermally driven property of the ELP. The ELP tag purification process is simple and inexpensive, and involves cycling the protein above and below the transition temperature of the ELP fusion followed by centrifugation to obtain the desired protein, without any need for chromatography. Consequently, using ELPs as a purification tag should be potentially interesting to biopharmaceutical companies who spend a significant percentage of their manufacturing costs on expensive protein purification techniques such as chromatography and filtration. However, ELP tags have not yet been used for commercial protein purification due to some challenges of translating this technique, which has been demonstrated mostly in academic laboratories, to a biotechnology manufacturing environment. The article first reviews the state-of-the-art in protein "ELPylation," and discusses some applications which have benefitted from using ELP as a fusion tag. Then, the article discusses the main advantages of using ELP as a purification tag, and evaluates the remaining hurdles for its implementation in industrial protein production. Biotechnol. Bioeng. 2016;113: 1617-1627. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

The effect of column purification on cDNA indirect labelling for microarrays

PubMed Central

Molas, M Lia; Kiss, John Z

2007-01-01

Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays. PMID:17597522

The effect of column purification on cDNA indirect labelling for microarrays.

PubMed

Molas, M Lia; Kiss, John Z

2007-06-27

The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.

Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.

PubMed

Akduman, Begüm; Uygun, Murat; Uygun, Deniz Aktaş; Akgöl, Sinan; Denizli, Adil

2013-12-01

In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase. © 2013 Elsevier B.V. All rights reserved.

Oriented immobilized anti-hIgG via F(c) fragment-imprinted PHEMA cryogel for IgG purification.

PubMed

Bereli, Nilay; Ertürk, Gizem; Tümer, M Aşkin; Say, Ridvan; Denizli, Adil

2013-05-01

Antibodies are used in many applications, especially as diagnostic and therapeutic agents. Among the various techniques used for the purification of antibodies, immunoaffinity chromatography is by far the most common. For this purpose, oriented immobilization of antibodies is an important step for the efficiency of purification step. In this study, F(c) fragment-imprinted poly(hydroxyethyl methacrylate) cryogel (MIP) was prepared for the oriented immobilization of anti-hIgG for IgG purification from human plasma. Non-imprinted poly(hydroxyethyl methacrylate) cryogel (NIP) was also prepared for random immobilization of anti-hIgG to compare the adsorption capacities of oriented (MIP/anti-hIgG) and random (NIP/anti-hIgG) cryogel columns. The amount of immobilized anti-hIgG was 19.8 mg/g for the NIP column and 23.7 mg/g for the MIP column. Although the amount of immobilized anti-hIgG was almost the same for the NIP and MIP columns, IgG adsorption capacity was found to be three times higher than the NIP/anti-hIgG column (29.7 mg/g) for the MIP/anti-hIgG column (86.9 mg/g). Higher IgG adsorption capacity was observed from human plasma (up to 106.4 mg/g) with the MIP/anti-hIgG cryogel column. Adsorbed IgG was eluted using 1.0 M NaCl with a purity of 96.7%. The results obtained here are very encouraging and showed the usability of MIP/anti-hIgG cryogel prepared via imprinting of Fc fragments as an alternative to conventional immunoaffinity techniques for IgG purification. Copyright © 2012 John Wiley & Sons, Ltd.

Dynamic pH junction high-speed counter-current chromatography coupled with microwave-assisted extraction for online separation and purification of alkaloids from Stephania cepharantha.

PubMed

Yuan, Zhiquan; Xiao, Xiaohua; Li, Gongke

2013-11-22

A simple and efficient dynamic pH junction high-speed counter-current chromatography method was developed and further applied to the online extraction, separation and purification of alkaloids from Stephania cepharantha by coupling with microwave-assisted extraction. Mineral acid and organic base were added into the mobile phase and the sample solution, respectively, leading to the formation of a dynamic pH junction in the column and causing focus of alkaloids. Selective focus of analytes can be achieved on the basis of velocity changes of the pH junction through appropriate selection of solvent systems and optimization of additive concentrations. The extract can be directly introduced into the HSCCC for the online extraction, separation and purification of alkaloids from S. cepharantha. Continuous separation can be easily achieved with the same solvent system. Under the optimum conditions, 6.0 g original sample was extracted with 60 mL of the upper phase of hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v) containing 10% triethylamine under 50 °C and 400 W irradiation power for 10 min, the extracts were directly separated and purified by high-speed counter-current chromatography. A total of 5.7 mg sinomenine, 8.3mg 6,7-di-O-acetylsinococuline, 17.9 mg berbamine, 12.7 mg isotetrandrine and 14.6 mg cepharanthine were obtained with purities of 96.7%, 93.7%, 98.7%, 97.3% and 99.3%, respectively. The online method provides good selectivity to ionizable compounds and improves the separation and purification efficiency of the high-speed counter-current chromatography technique. It has good potential for separation and purification of effective compounds from natural products. Copyright © 2013 Elsevier B.V. All rights reserved.

Nickel-Salen supported paramagnetic nanoparticles for 6-His-target recombinant protein affinity purification.

PubMed

Rashid, Zahra; Ghahremanzadeh, Ramin; Nejadmoghaddam, Mohammad-Reza; Nazari, Mahboobeh; Shokri, Mohammad-Reza; Naeimi, Hossein; Zarnani, Amir-Hassan

2017-03-24

In this research, a simple, efficient, inexpensive, rapid and high yield method for the purification of 6×histidine-tagged recombinant protein was developed. For this purpose, manganese ferrite magnetic nanoparticles (MNPs) were synthesized through a co-precipitation method and then they were conveniently surface-modified with tetraethyl orthosilicate (TEOS) in order to prevent oxidation and form high density of hydroxyl groups. Next, the salen ligand was prepared from condensation reaction of salicylaldehyde and 3-aminopropyl (trimethoxy) silane (APTMS) in 1:1 molar ratio; followed by complexation with Ni(OAc) 2 .4H 2 O. Finally, the prepared Ni(II)-salen complex conjugated to silica coated MNPs and MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles were obtained. The functionalized nanoparticles were spherical with an average diameter around 70nm. The obtained MNPs had a saturation magnetization about 54 emu/g and had super paramagnetic character. These MNPs were used efficiently to enrich recombinant histidine-tagged (His-tagged) protein-A from bacterial cell lysate. In about 45min, highly pure His-tagged recombinant protein was obtained, as judged by SDS-PAGE analysis and silver staining. The amount of target protein in flow through and washing fractions was minimal denoting the high efficiency of purification process. The average capacity of the matrix was found to be high and about 180±15mgg -1 (protein/MnFe 2 O 4 @SiO 2 @Ni-Salen complex). Collectively, purification process with MnFe 2 O 4 @SiO 2 @Ni-Salen complex nanoparticles is rapid, efficient, selective and whole purification can be carried out in only a single tube without the need for expensive systems. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparing postdeposition reactions of electrons and radicals with Pt nanostructures created by focused electron beam induced deposition

PubMed Central

Spencer, Julie A; Barclay, Michael; Gallagher, Miranda J; Winkler, Robert; Unlu, Ilyas; Wu, Yung-Chien; Plank, Harald; McElwee-White, Lisa

2017-01-01

The ability of electrons and atomic hydrogen (AH) to remove residual chlorine from PtCl2 deposits created from cis-Pt(CO)2Cl2 by focused electron beam induced deposition (FEBID) is evaluated. Auger electron spectroscopy (AES) and energy-dispersive X-ray spectroscopy (EDS) measurements as well as thermodynamics calculations support the idea that electrons can remove chlorine from PtCl2 structures via an electron-stimulated desorption (ESD) process. It was found that the effectiveness of electrons to purify deposits greater than a few nanometers in height is compromised by the limited escape depth of the chloride ions generated in the purification step. In contrast, chlorine atoms can be efficiently and completely removed from PtCl2 deposits using AH, regardless of the thickness of the deposit. Although AH was found to be extremely effective at chemically purifying PtCl2 deposits, its viability as a FEBID purification strategy is compromised by the mobility of transient Pt–H species formed during the purification process. Scanning electron microscopy data show that this results in the formation of porous structures and can even cause the deposit to lose structural integrity. However, this phenomenon suggests that the use of AH may be a useful strategy to create high surface area Pt catalysts and may reverse the effects of sintering. In marked contrast to the effect observed with AH, densification of the structure was observed during the postdeposition purification of PtCx deposits created from MeCpPtMe3 using atomic oxygen (AO), although the limited penetration depth of AO restricts its effectiveness as a purification strategy to relatively small nanostructures. PMID:29234576

Regulation of protein multipoint adsorption on ion-exchange adsorbent and its application to the purification of macromolecules.

PubMed

Huang, Yongdong; Bi, Jingxiu; Zhao, Lan; Ma, Guanghui; Su, Zhiguo

2010-12-01

Ion-exchange chromatography (IEC) using commercial ionic absorbents is a widely used technique for protein purification. Protein adsorption onto ion-exchange adsorbents often involves a multipoint adsorption. In IEC of multimeric proteins or "soft" proteins, the intense multipoint binding would make the further desorption difficult, even lead to the destruction of protein structure and the loss of its biological activity. In this paper, DEAE Sepharose FF adsorbents with controllable ligand densities from 0.020 to 0.183 mmol/ml were synthesized, and then the effect of ligand density on the static ion-exchange adsorption of bovine serum albumin (BSA) onto DEAE Sepharose FF was studied by batch adsorption technique. Steric mass-action (SMA) model was employed to analyze the static adsorption behavior. The results showed that the SMA model parameters, equilibrium constant (K(a)), characteristic number of binding sites (υ) and steric factor (σ), increased gradually with ligand density. Thus, it was feasible to regulate BSA multipoint adsorption by modulating the ligand density of ion-exchange adsorbent. Furthermore, IEC of hepatitis B surface antigen (HBsAg) using DEAE Sepharose FF adsorbents with different ligand densities was carried out, and the activity recovery of HBsAg was improved from 42% to 67% when the ligand density was decreased from 0.183 to 0.020 mmol/ml. Taking the activity recovery of HBsAg, the purification factor and the binding capacity into account, DEAE Sepharose FF with a ligand density of 0.041 mmol/ml was most effective for the purification of HBsAg. Such a strategy may also be beneficial for the purification of macromolecules and multimeric proteins. Copyright © 2010 Elsevier Inc. All rights reserved.

Design and analysis of siloxanes removal by adsorption from landfill gas for waste-to-energy processes.

PubMed

Elwell, Anthony C; Elsayed, Nada H; Kuhn, John N; Joseph, Babu

2018-03-01

Separation of volatile methyl siloxanes from landfill gas using fixed adsorption beds was modeled with the objective of identifying appropriate technology and the economics associated with this purification step. A general adsorption model assuming plug flow and radial symmetry was developed and used to conduct a parametric sweep of 162 unique cases. The varied parameters were adsorbent type (activated carbon and silica gel), bed height (3.05-9.15 m/10-30 ft), inlet siloxane concentration (5-15 mg/m 3 ), moisture content (0-100% relative humidity at STP or RH), and siloxane tolerance limit (0.094-9.4 mg/m 3 ) that correlated to three distinct energy conversion technologies (electricity production using engines or fuels cells or catalytic conversion to liquid hydrocarbon fuels). Due to the detrimental effect of RH on siloxane absorption, the maximum allowable moisture content of LFG before purification is 50% RH and moisture removal processes are also required. The design calculations using a selected case study show that the adsorption bed height required needed for 6 months minimum breakthrough time for catalytic fuel production is twice that for engine applications. Fuel cell applications require 3 times the bed height compared to engine applications. However, the purification costs amounted to 94%, 16% and 52% of recovered product value for engine, liquefaction, and fuel cell applications, respectively indicating the need for a high value product to justify purification costs. The approaches and conclusions can be extended to specific process conditions for landfill gas purification and to other processes that use biogas produced from waste as a feedstock. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

Coban, T Abdül Kadir; Ciftçi, Mehmet; Küfrevioğlu, O Irfan

2002-05-01

In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) 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: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)(-1). The overall purification was about 58-fold. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method, at 340 nm. In order to control the purification of 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 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60 degrees C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

Application of volcanic ash particles for protein affinity purification with a minimized silica-binding tag.

PubMed

Abdelhamid, Mohamed A A; Ikeda, Takeshi; Motomura, Kei; Tanaka, Tatsuya; Ishida, Takenori; Hirota, Ryuichi; Kuroda, Akio

2016-11-01

We recently reported that the spore coat protein, CotB1 (171 amino acids), from Bacillus cereus mediates silica biomineralization and that the polycationic C-terminal sequence of CotB1 (14 amino acids), designated CotB1p, serves as a silica-binding tag when fused to other proteins. Here, we reduced the length of this silica-binding tag to only seven amino acids (SB7 tag: RQSSRGR) while retaining its affinity for silica. Alanine scanning mutagenesis indicated that the three arginine residues in the SB7 tag play important roles in binding to a silica surface. Monomeric l-arginine, at concentrations of 0.3-0.5 M, was found to serve as a competitive eluent to release bound SB7-tagged proteins from silica surfaces. To develop a low-cost, silica-based affinity purification procedure, we used natural volcanic ash particles with a silica content of ∼70%, rather than pure synthetic silica particles, as an adsorbent for SB7-tagged proteins. Using green fluorescent protein, mCherry, and mKate2 as model proteins, our purification method achieved 75-90% recovery with ∼90% purity. These values are comparable to or even higher than that of the commonly used His-tag affinity purification. In addition to low cost, another advantage of our method is the use of l-arginine as the eluent because its protein-stabilizing effect would help minimize alteration of the intrinsic properties of the purified proteins. Our approach paves the way for the use of naturally occurring materials as adsorbents for simple, low-cost affinity purification. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.

Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression hostmore » offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.« less

Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles.

PubMed

Nestola, Piergiuseppe; Peixoto, Cristina; Villain, Louis; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

2015-12-24

We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core-shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally. Copyright © 2015 Elsevier B.V. All rights reserved.

Production and purification of the multifunctional enzyme horseradish peroxidase

PubMed Central

Spadiut, Oliver; Herwig, Christoph

2014-01-01

The oxidoreductase horseradish peroxidase (HRP) is used in numerous industrial and medical applications. In this review, we briefly describe this well-studied enzyme and focus on its promising use in targeted cancer treatment. In combination with a plant hormone, HRP can be used in specific enzyme–prodrug therapies. Despite this outstanding application, HRP has not found its way as a biopharmaceutical into targeted cancer therapy yet. The reasons therefore lie in the present low-yield production and cumbersome purification of this enzyme from its natural source. However, surface glycosylation renders the recombinant production of HRP difficult. Here, we compare different production hosts for HRP and summarize currently used production and purification strategies for this enzyme. We further present our own strategy of glycoengineering this powerful enzyme to allow recombinant high-yield production in Pichia pastoris and subsequent simple downstream processing. PMID:24683473

Fibulin-1 purification from human plasma using affinity chromatography on Factor H-Sepharose

PubMed Central

DiScipio, Richard G.; Liddington, Robert C.; Schraufstatter, Ingrid U.

2016-01-01

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

A scalable method for O-antigen purification applied to various Salmonella serovars

PubMed Central

Micoli, F.; Rondini, S.; Gavini, M.; Pisoni, I.; Lanzilao, L.; Colucci, A.M.; Giannelli, C.; Pippi, F.; Sollai, L.; Pinto, V.; Berti, F.; MacLennan, C.A.; Martin, L.B.; Saul, A.

2014-01-01

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations. PMID:23142430

Ion-exchange chromatography purification of extracellular vesicles.

PubMed

Kosanović, Maja; Milutinović, Bojana; Goč, Sanja; Mitić, Ninoslav; Janković, Miroslava

2017-08-01

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

A scintillator purification plant and fluid handling system for SNO+

NASA Astrophysics Data System (ADS)

Ford, Richard J.

2015-08-01

A large capacity purification plant and fluid handling system has been constructed for the SNO+ neutrino and double-beta decay experiment, located 6800 feet underground at SNOLAB, Canada. SNO+ is a refurbishment of the SNO detector to fill the acrylic vessel with liquid scintillator based on Linear Alkylbenzene (LAB) and 2 g/L PPO, and also has a phase to load natural tellurium into the scintillator for a double-beta decay experiment with 130Te. The plant includes processes multi-stage dual-stream distillation, column water extraction, steam stripping, and functionalized silica gel adsorption columns. The plant also includes systems for preparing the scintillator with PPO and metal-loading the scintillator for double-beta decay exposure. We review the basis of design, the purification principles, specifications for the plant, and the construction and installations. The construction and commissioning status is updated.

Augmenting static and dynamic mechanical strength of carbon nanotube/epoxy soft nanocomposites via modulation of purification and functionalization routes.

PubMed

Billing, Beant Kaur; Dhar, Purbarun; Singh, Narinder; Agnihotri, Prabhat K

2018-01-03

A detailed experimental investigation was carried out to establish the relationship between CNT purification and functionalization routes and the average response of CNT/epoxy nanocomposites under static and dynamic loading. It was shown that the relative improvement in the mechanical properties of the epoxy matrix due to the addition of CNTs depends on the choice of purification and functionalization steps. A better dispersion of CNTs was recorded for the functionalized CNTs as compared to the oxidized and CVD grown CNTs. Moreover, tensile, 3-point bending and nanoDMA testing performed on nanocomposites processed with CVD-grown, oxidized and functionalized CNTs revealed that COOH functionalization after the oxidation of CNTs at 350 °C is the optimized processing route to harness the excellent properties of CNTs in CNT/epoxy nanocomposites.

[Research and development of a vehicle-mounted drinking water installation and its purification effect].

PubMed

Gao, Junhong; Wan, Hong; Kong, Wei; Yue, Hong

2012-01-01

To provide a suitable vehicle-mounted installation to solve the problem of drinking water in the wild. The vehicle-mounted drinking water installation, made up of pre-treatment unit, purification unit, box and VECU, was used to storage, transport and purify water in the wild. The effect of purification was detected by assembling the installation in the wild and observing the change of water turbidity, TDS, the number of total bacteria and coliform bacteria before and after the treatment of water sources. The wild water sources, such as river water, rainwater, well water and spring water could be purified, and the quality of the treated water could meet the requirement of Drinking Water Quality Standard of CJ94-2005. The vehicle-mounted drinking water installation is suitable for purifying water sources in the wild for drinking use.

Methods of cell purification: a critical juncture for laboratory research and translational science.

PubMed

Amos, Peter J; Cagavi Bozkulak, Esra; Qyang, Yibing

2012-01-01

Research in cell biology and the development of translational technologies are driven by competition, public expectations, and regulatory oversight, putting these fields at a critical juncture. Success in these fields is quickly becoming dependent on the ability of researchers to identify and isolate specific cell populations from heterogeneous mixtures accurately and efficiently. Many methods for cell purification have been developed, and each has advantages and disadvantages that must be considered in light of the intended application. Current cell separation strategies make use of surface proteins, genetic expression, and physics to isolate specific cells by phenotypic traits. Cell purification is also dependent on the cellular reagents available for use and the intended application, as these factors may preclude certain mechanisms used in the processes of labeling and sorting cells. Copyright © 2011 S. Karger AG, Basel.

Efficacy of Flocculating and Other Emergency Water Purification Tablets

DTIC Science & Technology

1993-05-01

iodine tablet {1). The deficiencies identified with iodine tablets included slaw kill of Giardia cysts at law temperatures, medicinal taste and odor ...and the fact that undissolved solids, color and odor in field water ~e not rerroved. A market search for a new emergency water purification...tablet, or compound , was undertaken. The tablet had to be cOITll!Ercially available, nondevelopmental and satisfy the new military requirements. The

Superhydrophobic coated apparatus for liquid purification by evaporative condensation

DOEpatents

Simpson, John T; McNeany, Steve R; Dinsmore, Thomas V; Hunter, Scott R; Ivanov, Ilia N

2014-03-11

Disclosed are examples of apparatuses for evaporative purification of a contaminated liquid. In each example, there is a first vessel for storing the contaminated fluid. The first vessel includes a surface coated with a layer of superhydrophobic material and the surface is at least partially in contact with the contaminated liquid. The contaminants do not adhere to the surface as the purified liquid evaporates, thus simplifying maintenance of the apparatus.

Effects of Average Signed Area Between Two Item Characteristic Curves and Test Purification Procedures on the DIF Detection via the Mantel-Haenszel Method

ERIC Educational Resources Information Center

Wang, Wen-Chung; Su, Ya-Hui

2004-01-01

In this study we investigated the effects of the average signed area (ASA) between the item characteristic curves of the reference and focal groups and three test purification procedures on the uniform differential item functioning (DIF) detection via the Mantel-Haenszel (M-H) method through Monte Carlo simulations. The results showed that ASA,…

Renewable Energy Production from DoD Installation Solid Wastes by Anaerobic Digestion

DTIC Science & Technology

2016-06-01

and purification of methane -rich biogas was conducted at the US Air Force Academy. Cost and performance of the technology with respect to renewable...SUBJECT TERMS Food waste, FOG, solid waste, anaerobic digestion, methane , biogas, biomethane, biogas purification, vehicle fuel, renewable energy...The project demonstrated the ability to digest these wastes in a controlled and predictable manner to maximize the generation of biogas, a methane

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.

A two-column flash chromatography approach to pyoverdin production from Pseudomonas putida GB1.

PubMed

Duckworth, Owen W; Markarian, Dawn S; Parker, Dorothy L; Harrington, James M

2017-04-01

Our knowledge of the biological and environmental reactivity of siderophores is limited by the difficulty and cost of obtaining reasonable quantities by purification or synthesis. In this note, we describe a modified procedure for the low-cost, mg-scale purification of pyoverdin-type siderophores using a dual-flash chromatography (reverse-phase absorption and size exclusion) approach. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Combined distillation and normal freezing to purify elements of groups II and VI

NASA Technical Reports Server (NTRS)

Holland, L. R.

1984-01-01

A practical system and its application to the purification of Te and Cd is described. Single crystals are grown directly in vitreous silica ampoules subsequently used for sealed Bridgman growth of (Hg-Cd)Te. The system also prepares the ampoules by heating in high vacuum. Purification of the elements is by the combined effect of distillation and normal freezing. Transport and segregation are discussed.

Water Collection Purification System: Identifying CF Capabilities and Requirements, and Assessing off-the-Shelf Purification Systems

DTIC Science & Technology

2006-08-01

hydrocarbons, salinity, mercury , arsenic, cyanide, mustard gas, and nerve agents. Field engineers and WFEs are in charge of the testing. Additional testing...and biological (microorganisms, viruses) or they can come from the sediments (iron and manganese oxides, sulphide and polysulfide colloids) (Stumm...They are classified as inorganic and organic compounds. Inorganic compounds are heavy metals (lead, mercury , nickel, cadmium), and come from

Quantitative interaction proteomics using mass spectrometry.

PubMed

Wepf, Alexander; Glatter, Timo; Schmidt, Alexander; Aebersold, Ruedi; Gstaiger, Matthias

2009-03-01

We present a mass spectrometry-based strategy for the absolute quantification of protein complex components isolated through affinity purification. We quantified bait proteins via isotope-labeled reference peptides corresponding to an affinity tag sequence and prey proteins by label-free correlational quantification using the precursor ion signal intensities of proteotypic peptides generated in reciprocal purifications. We used this method to quantitatively analyze interaction stoichiometries in the human protein phosphatase 2A network.

Purification of preparative quantities of group B Streptococcus type III oligosaccharides.

PubMed

Paoletti, L C; Johnson, K D

1995-06-30

Many bacterial capsular polysaccharides are regularly repeating units of oligosaccharides. Bacterial oligosaccharides have been used in neoglycoconjugate vaccines and as reagents in the study of specific antibody binding. Unfortunately, separation methods have not been adequate for the purification of preparative quantities of bacterial oligosaccharides. Here we describe a size-exclusion procedure that resulted in the resolution of group B Streptococcus type III oligosaccharides composed of 4-25 sugars.

Polymer Based Highly Parallel Nanoscopic Sensors for Rapid Detection of Chemical and Biological Threats

DTIC Science & Technology

2007-09-18

Xuliang Han, PI of Brewer Science, Inc. Subcontract Center for Applied Science & Engineering Missouri State University 901 South National Avenue...Science an effective post-growth purification procedure was developed to reduce the amount of impurities, and several characterization techniques were...CNTs) contain a wide range of impurities from the growth process. At Brewer Science an effective post-growth purification procedure was developed to

Coherent-state information concentration and purification in atomic memory

NASA Astrophysics Data System (ADS)

Herec, Jiří; Filip, Radim

2006-12-01

We propose a feasible method of coherent-state information concentration and purification utilizing quantum memory. The method allows us to optimally concentrate and purify information carried by many noisy copies of an unknown coherent state (randomly distributed in time) to a single copy. Thus nonclassical resources and operations can be saved, if we compare information processing with many noisy copies and a single copy with concentrated and purified information.

Life Cycle Evolution and Systematics of Campanulariid Hydrozoans

DTIC Science & Technology

2004-09-01

kit according to manufacturer’s protocol. Purified PCR product was cycle-sequenced using either Big Dye 2 or 3 sequencing chemistry (ABI), following...ethidium bromide and purified with PCR purification kits (Qiagen). Purified products were cycle- sequenced with either Big Dye 2 or 3 sequencing chemistry...PCR purification kit (Qiagen). The purified product was cycle-sequenced using Big Dye 2 sequencing chemistry (ABI) following the manufacturer’s

DOE Office of Scientific and Technical Information (OSTI.GOV)

Davis, R.; Tao, L.; Scarlata, C.

This report describes one potential conversion process to hydrocarbon products by way of catalytic conversion of lignocellulosic-derived hydrolysate. This model leverages expertise established over time in biomass deconstruction and process integration research at NREL, while adding in new technology areas for sugar purification and catalysis. The overarching process design converts biomass to die die diesel- and naphtha-range fuels using dilute-acid pretreatment, enzymatic saccharification, purifications, and catalytic conversion focused on deoxygenating and oligomerizing biomass hydrolysates.

Purification for the XENONnT dark matter experiment

NASA Astrophysics Data System (ADS)

Brown, Ethan; Xenon Collaboration

2017-01-01

The XENON1T experiment uses 3.5 tons of liquid xenon in a cryogenic detector to search for dark matter. Its upgrade, XENONnT, will similarly house 7.5 tons of liquid xenon. Operation of these large detectors requires continual purification of the xenon in an external purifier, and the need for less than part per billion level oxygen in the xenon, coupled with the large quantity of xenon to be purified, places high demands on the rate of flow through this purification system. Building on the success of the XENON10 and XENON100 experiments, XENON1T circulates gaseous xenon through heated getters at a rate of up to 100 SLPM, pushing commercial pumps to their limits moving this large quantity of gas without interruption for several years. Two upgrades are considered for XENONnT. A custom high-capacity magnetic piston pump based on the one developed for the EXO200 experiment has been scaled up to support the high demands of this much larger experiment. Additionally, a liquid phase circulation and purification system that purifies the cryogenic liquid directly is being developed, which takes advantage of the much smaller volumetric flow demands of liquid relative to gas. The implementation of both upgrades will be presented. Supported by the National Science Foundation.

Collecting, archiving and processing DNA from wildlife samples using FTA® databasing paper

PubMed Central

Smith, LM; Burgoyne, LA

2004-01-01

Background Methods involving the analysis of nucleic acids have become widespread in the fields of traditional biology and ecology, however the storage and transport of samples collected in the field to the laboratory in such a manner to allow purification of intact nucleic acids can prove problematical. Results FTA® databasing paper is widely used in human forensic analysis for the storage of biological samples and for purification of nucleic acids. The possible uses of FTA® databasing paper in the purification of DNA from samples of wildlife origin were examined, with particular reference to problems expected due to the nature of samples of wildlife origin. The processing of blood and tissue samples, the possibility of excess DNA in blood samples due to nucleated erythrocytes, and the analysis of degraded samples were all examined, as was the question of long term storage of blood samples on FTA® paper. Examples of the end use of the purified DNA are given for all protocols and the rationale behind the processing procedures is also explained to allow the end user to adjust the protocols as required. Conclusions FTA® paper is eminently suitable for collection of, and purification of nucleic acids from, biological samples from a wide range of wildlife species. This technology makes the collection and storage of such samples much simpler. PMID:15072582

[Study on the extraction process and macroporous resin for purification of Timosaponin B II].

PubMed

Liu, Yan-Ping; Ding, Yue; Zhang, Tong; Wang, Bing; Cai, Zhen-Zhen; Tao, Jian-Sheng

2013-06-01

To optimize the extraction process and macroporous resin for purification of Timosaponin B II from Anemarrhena asphodeloides. Orthogonal design L9 (34) was employed to optimize the circumfluence extraction conditions by taking the extraction yield of Timosaponin B II as index. The absorption-desorption characteristics of eight kinds of macroporous resins were evaluated, then the best resin was chosen to optimize the purification process conditions. The optimum extraction conditions were as follows: the herb was extracted for 2 times (2 hours each time) with 8.5-fold 50% ethanol at the first time and 6-fold 50% ethanol at the second time. HPD100 resin showed a good property for the absorption-desorption of Timosaponin B II. The optimum technological conditions of HPD100 resin were as follows:the solution concentration was 0.23 mg/mL, the amount of saturated adsorption at 4/5 body volumn (BV) resin, the HPD100 resin was washed with 3 BV water and 6 BV 20% ethanol solution to remove the impurity, then the Timosaponin B II was desorbed by 5 BV ethanol solution. The purity of Timosaponin B II was about 50%. The optimized extraction process and purification is stable, efficient and suitable for industrial production.