• Title/Summary/Keyword: G C Separation

Search Result 392, Processing Time 0.029 seconds

Expression of Polyhistidine-Containing Fusion Human HepG2 Type Glucose Transport Protein in Spodoptera Cells and Its Purification Using a Metal Affinity Chromatography

  • Lee, Chong-Kee
    • Biomedical Science Letters
    • /
    • v.16 no.3
    • /
    • pp.201-206
    • /
    • 2010
  • In order to develop procedures for the rapid isolation of recombinant sugar transporter in functional form from away from the endogenous insect cell transporter, gene fusion techniques were exploited. Briefly, BamH1-digested human HepG2 type glucose transport protein cDNA was first cloned into a transfer vector pBlueBacHis, containing a tract of six histidine residues. Recombinant baculoviruses including the human cDNA were then generated by allelic exchange following transfection of insect cells with wild-type BaculoGold virus DNA and the recombinant transfer vector. Plaque assay was then performed to obtain and purify recombinant viruses expressing the human transport protein. All the cell samples that had been infected with viruses from the several blue plaques exhibited a positive reaction in the immnuassay, demonstrating expression of the glucose transport protein. In contrast, no color development in the immunoassay was observed for cells infected with the wild-type virus or no virus. Immunoblot analysis showed that a major immunoreactive band of apparent Mr 43,000~44,000 was evident in the lysate from cells infected with the recombinant baculovirus. Following expression of the recombinant fusion protein with the metal-binding domain and enterokinase cleavage site, the fusion protein was recovered by competition with imidizole using immobilized metal charged resin. The leader peptide was then removed from the fusion protein by cleavage with porcine enterokinase. Final separation of the recombinant protein of the interest was achieved by passage over $Ni^{2+}$-charged resin under binding conditions. The expressed transport protein bound cytochalasin B and demonstrated a functional similarity to its human counterpart.

Characterization of a Toxin isolated from Bacillus thuringiensis var. alesti and Its Toxicity to the Silkworm, Bombyx mori (Bacillus thuringiensis var. alesti로부터 분리된 독소의 성상과 누에에 대한 독성)

  • 이영근;조용섭;임종성
    • Journal of Sericultural and Entomological Science
    • /
    • v.17 no.1
    • /
    • pp.55-62
    • /
    • 1975
  • In the studies of characterization of crystal prototoxin isolated from Bacillus thuringinensis var. alesti and its bioassay to the silkworm larvae, Bombyx mori L., the following results were obtained. 1. The complete lysis of bacteria grown on the nutrient agar at 30$^{\circ}C$ in an incubator took 30 days, but the period could be reduced by a half in a devised broth media in a fermentation jar. 2. The protein toxin extracted directly from a mixture of crystals and spores without separation of crystals and spores was pure as same as the protein toxin extracted from only crysals separated. 3. By SLS polyacrylamide gel electrophoresis of prototoxin released from crystals in alkali (pH 9.5) three different proteins in molecular weights of 120,000, 87,000, 74,000 were separated. 4. In the bioassay of the toxin to the silkworm larvae, LD$\sub$50/ per gram of the larvae in the 4th instar was 0.080 $\mu\textrm{g}$.

  • PDF

Quantitative Determination of the Thirteen Marker Components in Banhasasim-Tang Decoction Using an Ultra-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry (LC-MS/MS를 이용한 반하사심탕 물 추출물 중 13종 성분의 함량분석)

  • Seo, Chang-Seob;Shin, Hyeun-Kyoo
    • Korean Journal of Pharmacognosy
    • /
    • v.47 no.1
    • /
    • pp.62-72
    • /
    • 2016
  • Banhasasim-tang is a well-known traditional Korean herbal formula and has been used clinically for the treatment of gastric disease, including acute and chronic gastritis, diarrhea and gastric ulcers in Korea. In this study, an ultra-performance liquid chromatography-electrospray ionization-mass spectrometer method was developed for the quantitative determination of the 13 marker constituents, homogentisic acid (1), 3,4-dihydroxybenzaldehyde (2), spinosin (3), liquiritin (4), baicalin (5), ginsenoside Rg1 (6), liquiritigenin (7), wogonoside (8), ginsenoside Rb1 (9), baicalein (10), glycyrrhizin (11), wogonin (12), and 6-gingerol (13) in Banhasasim-tang decoction. Separation of the compounds 1-13 was using an UPLC BEH $C_{18}$ ($100{\times}2.1mm$, $1.7{\mu}m$) column and column oven temperature was maintained at $45^{\circ}C$. The mobile phase consisted of 0.1% (v/v) formic acid in water (A) and acetonitrile (B) by gradient elution. The injection volume and flow rate were $2.0{\mu}L$ and 0.3 mL/min, respectively. Calibration curves of the compounds 1-13 were showed with $r^2$ values ${\geq}0.9908$. The limit of detection and limit of quantification values of the compounds 1-13 were 0.04-1.11 ng/mL and 0.13-3.33 ng/mL, respectively. Among the these compounds, the compounds 1-3 were not detected, while the compounds 4-13 were detected in the ranges of $3.20-107,062.98{\mu}g/g$ in Banhasasim-tang sample.

Quantitative Analysis of the Eleven Marker Components in Traditional Korean Formula, Jakyakgamcho-Tang Decoction Using an Ultra-Performance Liquid Chromatography Coupled to Electrospray Ionization Tandem Mass Spectrometry (UPLC-MS/MS를 이용한 작약감초탕 물 추출물 중 11종 성분의 함량분석)

  • Seo, Chang-Seob;Shin, Hyeun-Kyoo
    • YAKHAK HOEJI
    • /
    • v.60 no.2
    • /
    • pp.64-72
    • /
    • 2016
  • Jakyakgamcho-tang is a well-known traditional herbal medicine and has been used for the treatment of mainly pains in oriental medicine. In this study, analytical method for the quantitative determination of the eleven marker components, gallic acid (1), oxypaeoniflorin (2), paeoniflorin (3), albiflorin (4), liquiritin (5), isoliquiritin (6), ononin (7), liquiritigenin (8), benzoylpaeoniflorin (9), paeonol (10), and glycyrrhizin (11) in Jakyakgamcho-tang decoction was performed using an ultra-performance liquid chromatography-electrospray ionization-mass spectrometer. The analytical column for separation of the compounds 1~11 was used an UPLC BEH $C_{18}$ ($100{\times}2.1mm$, $1.7{\mu}m$) column and column oven temperature was maintained at $45^{\circ}C$. The mobile phase consisted of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B) by gradient elution. The flow rate was 0.3 ml/min and injection volume was $2.0{\mu}l$. Correlation coefficient in the calibration curves of the compounds 1~11 were showed a good linearity with more than 0.99. The limit of detection and limit of quantification values of the compounds 1~13 were detected in the ranges 0.06~18.43 ng/ml and 0.18~58.29 ng/ml, respectively. Among the compounds 1~11, the compounds 10 were not detected in this sample, while the ten compounds, 1~9 and 11, were detected $44.05{\sim}19,289.05{\mu}g/g$ in Jakyakgamcho-tang extract.

Qualitative and Quantitative Analysis of Thirteen Marker Components in Traditional Korean Formula, Samryeongbaekchul-san using an Ultra-Performance Liquid Chromatography Equipped with Electrospray Ionization Tandem Mass Spectrometry

  • Seo, Chang-Seob;Shin, Hyeun-Kyoo
    • Natural Product Sciences
    • /
    • v.22 no.2
    • /
    • pp.93-101
    • /
    • 2016
  • For efficient quality control of the Samryeongbaekchul-san decoction, a powerful and accurate an ultra-performance liquid chromatography (UPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS) method was developed for quantitative analysis of the thirteen constituents: allantoin (1), spinosin (2), liquiritin (3), ginsenoside Rg1 (4), liquiritigenin (5), platycodin D2 (6), platycodin D (7), ginsenoside Rb1 (8), glycyrrhizin (9), 6-gingerol (10), atractylenolide III (11), atractylenolide II (12), and atractylenolide I (13). Separation of the compounds 1 - 13 was performed on a UPLC BEH $C_{18}$ column ($2.1{\times}100mm$, $1.7{\mu}m$) at a column temperature of $40^{\circ}C$ with a gradient solvent system of 0.1% (v/v) formic acid aqueous-acetonitrile. The flow rate and injection volume were 0.3 mL/min and $2.0{\mu}L$. Calibration curves of all compounds were showed good linearity with values of the correlation coefficient ${\geq}0.9920$ within the test ranges. The values of limits of detection and quantification for all analytes were 0.04 - 4.53 ng/mL and 0.13 - 13.60 ng/mL. The result of an experiment, compounds 2, 6, 12, and 13 were not detected while compounds 1, 3 - 5, and 7 - 11 were detected with 1,570.42, 5,239.85, 299.35, 318.88, 562.27, 340.87, 12,253.69, 73.80, and $115.01{\mu}g/g$, respectively.

Breeding Process and Characteristics of Gopoong, a New Variety of Panax ginseng C.A. Meyer. (인삼(Panax ginseng C.A. Meyer) 신품종 고풍의 육성경과 및 생육특성)

  • Kwon, Woo-Saeng;Lee, Jang-Ho;Park, Chan-Soo;Yang, Deok-Chun
    • Journal of Ginseng Research
    • /
    • v.27 no.2
    • /
    • pp.86-91
    • /
    • 2003
  • To develop a new ginseng variety with good quality and high yielding, a lot of individual ginseng plant were selected in the farmers'fields in 1968. Among them, a promising line,680-83-4, has been developed through comparative cultivation of several lines selected with pure line separation from local races in KT&C Central Research Institute, preliminary and advanced yield trials were performed fir 8 years.680-83-4 was designated as KG103, which was then registered as a new variety “Gopoong” with the regional yield and adaptation trials for 10 years (1981-1990) on December 27,2000 in Korea. Gopoong has different phenotype of a dark violet stem and dark red fruit and inverted triangle shape of berries cluster as compared with other varieties. Taproot of Gopoong was longer than local race Jakyungjong, and root yield was 4.5% higher than local race Jakyungjong. In red ginseng quality, the rates of Chun-Jeesam grade(Chun and Jee means 1 st and 2nd grade, respectively) were 16.6% and 9.4% for Gopoong and Jakyungjong, respectively. In these results, it was that Gopoong was superior ginseng line with good quality far manufacture of red ginseng.

Separation and Characterization of Endo-Polygalacturonase from Aspergillus niger (Aspergillus niger가 생산(生産)하는 Endo-Polygalacturonase의 분리(分離)와 특성(特性))

  • Park, Kyong-Bin;Park, Kwan-Hwa
    • Korean Journal of Food Science and Technology
    • /
    • v.16 no.1
    • /
    • pp.41-46
    • /
    • 1984
  • The pectic enzymes produced from Aspergillus niger were separated into three fractions (F-A, F-I and F-II) by means of Sephadex and DEAF-Sephadex column chromatography. Each enzyme fraction was characterized by determining viscosity change and reducing surgar of the pectic acid-enzyme mixture and analyzing thin layer chromatogram of the reaction products. F-I rapidly reduced the viscosity of pectic acid solution and released reducing groups in a random manner so that appeared to be an endo-polygalacturonase (endo-PG). The optimum pH of endo-PG for viscosity reducing activity was 4.2 and that for releasing reducing surgar was 4.7. In the thermal inactivation of endo-PG of $30-45^{\circ}C$, the enthalpy of activation was 217.3 kj/mole and z-value was $7.5^{\circ}C$. F-II and F-A were determined as endo-polymethylgalacturonase and exo-polygalacturonase, respectively.

  • PDF

Sequential Separation of Lysozyme and Ovalbumin from Chicken Egg White

  • Abeyrathne, Nalaka Sandun;Lee, Hyun Yong;Ahn, Dong Uk
    • Food Science of Animal Resources
    • /
    • v.33 no.4
    • /
    • pp.501-507
    • /
    • 2013
  • Lysozyme was trapped from $2{\times}$ diluted egg white using Amberlite FPC 3500 ion exchange resin (1 g/10mL of egg white). The lysozyme bound to the resin was recovered using 0.1 N glycine-NaOH buffers, pH 9.0, containing 0.5 M NaCl. After separating lysozyme, the pH of the egg white solution was adjusted to 4.75 and centrifuged to remove interfering proteins. The supernatant was collected, added with 2.5% citric acid and 5.0% ammonium sulfate combination to precipitate egg white proteins, except for ovalbumin. After centrifugation, both supernatant (S1) and precipitant were collected. The precipitant was dissolved with 4 volumes of distilled water, and then 2.0% ammonium sulfate and 1.5% citric acid combinations added, stirred overnight in a cold room, and centrifuged. The resulting supernatant (S2) was pooled with the first supernatant (S1), desalted using an ultrafiltration unit, heat-treated at $70^{\circ}C$ for 15 min, and then centrifuged. The supernatant was collected as an ovalbumin fraction and lyophilized. The separated proteins were confirmed using Western blotting. The yield of lysozyme and ovalbumin was > 88.9% and > 97.7%, respectively, and the purity of lysozyme and ovalbumin was > 97% and 87%, respectively. The results indicated that the protocol was simple, and separated lysozyme and ovalbumin effectively.

Readeveloping Turbulent Boundary Layer after Separation-Reattachment(I) (박리-재부착 이후의 재발달 난류경계층 I)

  • 백세진;유정열
    • Transactions of the Korean Society of Mechanical Engineers
    • /
    • v.13 no.4
    • /
    • pp.780-788
    • /
    • 1989
  • An experimental study has been performed to investigate the process from nonequilibrium state to equilibrium state in redeveloping turbulent boundary layer beyond separation-reattachment using pitot tube and hot-wire anemometer. The model sued in the experiment has the form of a backward facing step which is assembled by a two-dimensional 4:1 half elipse and a plate. Measurements are carried out up to a distance of about 50 step height downstream of the step, where the reattachment observed at about x/h=6.5. The profiles of the shape factor H the Clauser parameter G and the coefficient of friction $C^{f}$ exhibited the characteristics similar to those of the equilibrium turbulent boundary layer from x/h=25, and the profiles of the trubulent quantities did from x/h=35. However, the wake region of the boundary layer does not seem to recover the equilibrium turbulent boundary layer even at x/h=50. By considering the distributions of the intermittency factor it has been noted that the turbulence structure changes gradually from a mixing layer to a turbulent boundary layer along downstream direction after reattachment. This becomes clearer as we analyse the one-dimensional energy spectra and the dissipation energy spectra which are measured and caculated at various downstream positions after the backward facing step.p.

3D Numerical investigation of a rounded corner square cylinder for supercritical flows

  • Vishwanath, Nivedan;Saravanakumar, Aditya K.;Dwivedi, Kush;Murthy, Kalluri R.C.;Gurugubelli, Pardha S.;Rajasekharan, Sabareesh G.
    • Wind and Structures
    • /
    • v.35 no.1
    • /
    • pp.55-66
    • /
    • 2022
  • Tall buildings are often subjected to steady and unsteady forces due to external wind flows. Measurement and mitigation of these forces becomes critical to structural design in engineering applications. Over the last few decades, many approaches such as modification of the external geometry of structures have been investigated to mitigate wind-induced load. One such proven geometric modification involved the rounding of sharp corners. In this work, we systematically analyze the impact of rounded corner radii on the reducing the flow-induced loading on a square cylinder. We perform 3-Dimensional (3D) simulations for high Reynolds number flows (Re=1 × 105) which are more likely to be encountered in practical applications. An Improved Delayed Detached Eddy Simulation (IDDES) method capable of capturing flow accurately at large Reynolds numbers is employed in this study. The IDDES formulation uses a k-ω Shear Stress Transport (SST) model for near-wall modelling that prevents mesh-induced separation of the boundary layer. The effects of these corner modifications are analyzed in terms of the resulting variations in the mean and fluctuating components of the aerodynamic forces compared to a square cylinder with no geometric changes. Plots of the angular distribution of the mean and fluctuating coefficient of pressure along the square cylinder's surface illustrate the effects of corner modifications on the different parts of the cylinder. The windward corner's separation angle was observed to decrease with an increase in radius, resulting in a narrower and longer recirculation region. Furthermore, with an increase in radius, a reduction in the fluctuating lift, mean drag, and fluctuating drag coefficients has been observed.