• Title/Summary/Keyword: Eluate

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Cloning, Sequencing, and Expression of cDNA Encoding Bovine Prion Protein

  • Kang, Sang-Gyun;Kang, Sung-Keun;Lee, Deog-Yong;Park, Yong-Ho;Hwang, Woo-Suk;Yoo, Han-Sang
    • Journal of Microbiology and Biotechnology
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    • v.14 no.2
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    • pp.417-421
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    • 2004
  • A normal prion protein (PrPc) is converted to a protease resistant isoform (PrPsc) by an apparent self-propagating activity in bovine spongiform encephalopathies (BSE), which is a neurodegenerative disease. The cDNA encoding bovine PrP open reading frame (ORP) in Korean cattle was cloned by polymerase chain reaction (PCR). The cloned cDNA had a length of 795 base pairs which coded for a protein of 264 amino acid residues with a calculated molecular mass of 28.6 kDa. Identities of 90, 90, 79 and 78% on nucleotide and 94, 94, 84, and 84% on amino acid sequence were shown to PrP genes from sheep, goat, human, and mouse, respectively. The cloned DNA was ligated into the pQE30 expression vector and transformed into E. coli M15. The PrP was expressed by induction with isopropyl-$\beta$-D-thiogalactoside (IPTG) and purified on the Ni-NTA affinity column. High specific activities of the recombinant PrP were observed in the fraction of pH 5.8 eluate and showed a molecular mass of-29 kDa on SDS-PAGE and Western blot analysis.

Quantitative Analysis of Cinnamaldehyde, Cinnamylalcohol and Salicylaldehyde in Commercial Biopesticides Containing Cinnamon Extract Using Gas Chromatography - Flame Ionization Detector

  • Lim, Sung-Jin;Lee, Ji-Hye;Kim, Jin-Hyo;Choi, Geun-Hyoung;Cho, Nam-Jun;Park, Byung-Jun
    • Korean Journal of Environmental Agriculture
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    • v.33 no.3
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    • pp.213-219
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    • 2014
  • BACKGROUND: In an environment-friendly agriculture, plant extracts have been perceived as alternatives of synthetic pesticides. The Environment-friendly Agriculture Promotion Act of Korea has approved cinnamon extract as a matter for the production of commercial biopesticides. Thirteen commercial biopesticides containing cinnamon extract have been marketed locally. However, the analytical method for the quality control of these biopesticides containing cinnamon extract has not been studied. METHODS AND RESULTS: Cartridge clean-up method for the determination of cinnamaldehyde, cinnamylalcohol and salicylaldehyde in biopesticides containing cinnamon extract was developed and validated by gas chromatography (GC). The clean-up method was optimized with HLB SPE cartridges for the bioactive substance in biopesticides containing cinnamon extract, and the eluate was analyzed by GC. The developed method was validated, and the LOQ and recovery rates of cinnamaldehyde, cinnamylalcohol and salicylaldehyde were 0.139, 0.067 and $0.062mgL^{-1}$ and 84.2, 86.5 and 82.1%, respectively. The contents of cinnamaldehyde, cinnamylalcohol and salicylaldehyde were analyzed using the developed method in the 13 commercial biopesticides. Results showed 0.06-17.37%,

Determination of Forward Dissolution Rate of Glass by a Single-Pass Flow-Through Test (Single-Pass Flow-Through Test방법에 의한 유리의 정용해율 측정)

  • Kim Seung-Soo;Chun Kwan-Sik;Choi Jong-Won;Kim Sung-Ki;Hahn Pil-Soo
    • Journal of Nuclear Fuel Cycle and Waste Technology(JNFCWT)
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    • v.3 no.4
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    • pp.335-340
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    • 2005
  • The forward dissolution rate of a borosilicate waste glass was determined as an interlaboratory study(ILS) testing program for the evaluation of precision in the measurement of the dissolution rate or a waste glass using a single-pass flow-through(SPFT) test, whose conducting practice has been written for standardization through American Society for Testing and Materials (ASTM). A simulated low-activity waste glass powder with a size of 100/200 mesh was dissolved by lithium buffer solution (pH=10) at 70? under Ar atmosphere. By plotting the dissolution rates as a function of silicon and boron concentration in eluate, the forward dissolution rate of the glass was obtained as about $2.7\times10^{-5}g{\cdot}m{\cdot}s^{-1}$ in our laboratory.

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Solid-phase Refolding of Poly-lysine Tagged Fusion Protein of hEGF and Angiogenin

  • Park Sang Joong;Ryu Kang;Suh Chang Woo;Chai Young Gyu;Kwon Oh Byung;Park Seung Kook;Lee Eun Kyu
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.7 no.1
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    • pp.1-5
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    • 2002
  • A fusion protein, consisting of a human epidermal growth factor (hEGF) as the recognition domain and human angiogenin as the toxin domain, can be used as a targeted therapeutic against breast cancer cells among others. The fusion protein was expressed as inclusion body in recombinant E. coli, and when the conventional, solution-phase refolding process was used the refolding yield was very low due to severe aggregation. It was probably because of the opposite electric charge at a neutral pH resulting from the vastly different pI values of each domain. The solid-phase refolding process that exploited the ionic interactions between ionic exchanger surface and the fusion protein was tried, but the adsorption yield was also very low, below $ 30\%$, regardless of the resins and pH conditions used. Therefore, to provide a higher ionic affinity toward the solid matrix, six lysine residues were tagged to the N-terminus of the hEGF domain. When heparin-Sepharose was used as the matrix, the adsorption capacity increased 2.5-3 times to about $88\%$. Besides the intrinsic affinity of angiogenin to heparin, the poly-lysine tag provided additional ionic affinity. And the subsequent refolding yield increased nearly 13-fold, from ca. $4.8\%$ in the conventional refolding of the untagged fusion protein to $63.6\%$. The process was highly reproducible. The refolded protein in the column eluate retained RNase bioactivity of angiogenin.

Matrix solid phase dispersion(MSPD) extraction and HPLC determination of enrofloxacin and ciprofloxacin in pork muscle tissue (시료고체상분산(matrix solid phase dispersion)전처리법과 액체크로마토그라피를 이용한 돈육중 enrofloxacin 및 ciprofloxacin 분석)

  • Kang, Hwan-goo;Son, Seong-wan;Lee, Hye-sook;Kim, Jae-hak;Cho, Myung-haing
    • Korean Journal of Veterinary Research
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    • v.37 no.1
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    • pp.195-202
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    • 1997
  • A method for the isolation by matrix solid phase dispersion method and liquid chromatographic determination of enrofloxacin and ciprofloxacin in pork muscle tissue is presented. Blank or enrofloxacin and ciprofloxacin spiked samples(0.5g) containing 0.05g oxalic acid were blended with $C_{18}$(octadecylsilyl derivatized silica) packing material. After homogenization, $C_{18}$/muscle tissue matrix was transferred to glass column made from 10ml glass syringe and filter paper, and compressed to 4~4.5ml volume. A column was washed with 8ml of hexane and dried under vacuum. Interfering materials were removed by ethylacetate 8ml and dried, following which enrofloxacin and ciprofloxacin were eluted with 8ml of methanal under gravity. The eluate containing enrofloxacin and ciprofloxacin wase free from interfering compound when analysed by HPLC with UV detection at 278nm. Enrofloxacin and ciprofloxacin showed linear response with UV detector at the range of $0.05{\sim}1.0{\mu}g/ml$ and eluted within 5ml elution volume of methanol from the matrix. Fortified sample containing 0.05g oxalic acid represented more good recoveries than that of control sample. Average percentages of enrofloxacin and ciprofloxacin were $93.30{\pm}4.56%$ and $91.84{\pm}4.17%$, respectively, for the concentration range(0.05, 0.1, 0.25, 0.5 and $0.75{\mu}g/g$). The interassay variability of enrofloxacin was $6.02{\pm}5.33%$ with an intra-assay variability of 4.89% and $6.75{\pm}2.68%$ with 4.54% for ciprofloxacin. Detection limit of enrofloxacin and ciprofloxacin was $0.030{\mu}g/g$ in the spiked sample.

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Effects of four novel root-end filling materials on the viability of periodontal ligament fibroblasts

  • Akbulut, Makbule Bilge;Arpaci, Pembegul Uyar;Eldeniz, Ayce Unverdi
    • Restorative Dentistry and Endodontics
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    • v.43 no.3
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    • pp.24.1-24.12
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    • 2018
  • Objectives: The aim of this in vitro study was to evaluate the biocompatibility of newly proposed root-end filling materials, Biodentine, Micro-Mega mineral trioxide aggregate (MM-MTA), polymethylmethacrylate (PMMA) bone cement, and Smart Dentin Replacement (SDR), in comparison with contemporary root-end filling materials, intermediate restorative material (IRM), Dyract compomer, ProRoot MTA (PMTA), and Vitrebond, using human periodontal ligament (hPDL) fibroblasts. Materials and Methods: Ten discs from each material were fabricated in sterile Teflon molds and 24-hour eluates were obtained from each root-end filling material in cell culture media after 1- or 3-day setting. hPDL fibroblasts were plated at a density of $5{\times}10^3/well$, and were incubated for 24 hours with 1:1, 1:2, 1:4, and 1:8 dilutions of eluates. Cell viability was evaluated by XTT assay. Data was statistically analysed. Apoptotic/necrotic activity of PDL cells exposed to material eluates was established by flow cytometry. Results: The Vitrebond and IRM were significantly more cytotoxic than the other root-end filling materials (p < 0.05). Those cells exposed to the Biodentine and Dyract compomer eluates showed the highest survival rates (p < 0.05), while the PMTA, MM-MTA, SDR, and PMMA groups exhibited similar cell viabilities. Three-day samples were more cytotoxic than 1-day samples (p < 0.05). Eluates from the cements at 1:1 dilution were significantly more cytotoxic (p < 0.05). Vitrebond induced cell necrosis as indicated by flow cytometry. Conclusions: This in vitro study demonstrated that Biodentine and Compomer were more biocompatible than the other root-end filling materials. Vitrebond eluate caused necrotic cell death.

Quantitative Analysis of Rotenone and Deguelin in Biopesticides Containing Derris Extract by Ultra performance Liquid Chromatography (UPLC를 활용한 데리스 추출물 함유 유기농자재 중 Rotenone과 Deguelin 정량분석)

  • Lim, Sung-Jin;Kim, Jin-Hyo;Choi, Geun-Hyoung;Park, Byung-Jun
    • Korean Journal of Environmental Agriculture
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    • v.34 no.1
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    • pp.52-56
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    • 2015
  • BACKGROUND: Three commercial biopesticides containing derris extract, which is permitted as a commercial biopesticide substances by the Environmentally-friendly Agriculture Promotion Act, have been marketed in Korea. But, the quantitative analytical method of active substances for crop protection in biopesticides containing derris extract has not known. METHODS AND RESULTS: Solid phase extraction (SPE) cartridge clean-up method for the quantitative analysis of rotenone and deguelin in biopesticides containing derris extract was developed and validated by ultra-performance liquid chromatography (UPLC). The clean-up method was established using hydrophilic lipophilic balance (HLB) SPE cartridges for the bioactive substances in biopesticides containing derris extract, and the eluate was analyzed to quantify the rotenone and deguelin by the UPLC. LOQ and recovery rates of rotenone and deguelin were 0.085 and 0.044 mg/L, 95.7 and 93.3%, respectively. The content of rotenone and deguelin in three biopesticides containing derris extract were analyzed by the developed method, the results showed 0.001-0.236 and

A rapid separation of Cs, Sr and Ba using gas pressurized extraction chromatography with inductively coupled plasma-mass spectrometry

  • Sojin Jeong;Jihye Kim;Hanul Cho;Hwakyeung Jeong;Byungman Kang;Sang Ho Lim
    • Analytical Science and Technology
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    • v.37 no.2
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    • pp.123-129
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    • 2024
  • We present a rapid method for the determination of Cs, Sr, and Ba, heat generators found in highly active liquid wastes, by gas-pressurized extraction chromatography (GPEC) using a column containing a cation-exchange resin. GPEC is a microscale column chromatographic technique that uses a constant flow rate of solvent (0.07 mL/min) with pressurized nitrogen gas supplied through a valve. In particular, because this method uses a small sample volume (a few hundred microliters), it produces less chemical waste and allows for faster separation compared to traditional column chromatography. In this study, we evaluated the separation of Cs, Sr, and Ba using GPEC. The eluate from the column (GPEC or conventional column chromatography) was quantitatively analyzed using inductively coupled plasma-mass spectrometry to measure the column recovery and precision. The column reproducibility of the proposed GPEC system (RSDs of recoveries) ranged from 2.7 to 4.1 %, and the column recoveries for the three elements ranged from 72 to 98% when aqueous HCl was used as the eluent. The GPEC results are slightly different in efficiency and separation resolution compared to those of conventional column chromatography because of the differences in the eluent flow rate as well as the internal diameter and length of the column. However, the two methods had similar recoveries for Cs and Sr, and the precision of GPEC was improved by two-fold. Remarkably, the solvent volume required for GPEC analysis was five times lower than that of the conventional method, and the total analysis time was 11 times shorter.

Simultaneous Determination of Ultra-Trace Pesticides and Synthetic Materials in Surface Water by LC-ESI-MS/MS (하천수에서 LC-ESI-MS/MS에 의한 극미량 농약류 및 합성원료의 동시분석법)

  • Hong, Seon-Haw;Lee, Jun-Bae;Lee, Soo-Hyung;Cho, Young-Hwan;Shin, Ho-Sang
    • Journal of the Korean Chemical Society
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    • v.59 no.3
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    • pp.225-232
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    • 2015
  • A liquid chromatography-electrospray ionization-tandem mass spectrometry method (LC-ESI-MS/MS) was used for determining seven pesticides (2,4-dichlorophenoxyacetic acid, methomyl, aldicarb, 2-methyl- 4-chlorophenoxy- acetic acid, molinate, carbaryl and carbofuran) and two synthetic materials (quinoline and bisphenol-A) in surface water. The analytes were extracted using solid-phase extraction (SPE). The eluate was concentrated by nitrogen gas. 100 microliters of 30% (v/v) methanol aqueous solution were used to dissolve the residue and an aliquot of the reconstituted solution was directly injected into LC-ESI-MS/MS after the filtration using 0.2 μm polytetrafluoroethylene (PTFE) syringe filter. Under the established condition, the calibration curves of the analytes were linear with correlation coefficients of above 0.997. The quantification limit was 0.002~0.011 μg/L and the relative standard deviations were less than 16.4%. In addition, accuracy was in the range of 84~107% and the recoveries were values between 56.2 and 98.6%. In this study, the developed method was applied to the analysis of real surface water samples.

A Study on the pH Characteristic of Recycle Aggregate According to Test Methods and Elapsed Time (측정방법 및 시간경과에 따른 순환골재의 pH 특성에 관한 연구)

  • Song, Tae-Hyeob;Lee, Jong-Chan;Lee, Sae-Hyun
    • Journal of the Korean Recycled Construction Resources Institute
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    • v.6 no.3
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    • pp.61-68
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    • 2011
  • The pH of recycled aggregate is over 12 because of high alkalinity of cement. This high alkalinity give circumstance harmful effect. The high alkalinity of recycled aggregate can appear variously according to aggregate properties, elution time of alkalinity, aggregate size, and so on. This study analyzed recycled aggregate properties according to wet and dry type manufacture process; different test methods: waste official test, soil contamination official test, BS EN 1744-3 standard; elution time and different size to test effects of various condition. These test results can contribute to solving environmental problems by recycled aggregate. In the test results, pH of recycled aggregate was as higher as smaller particle size and as time elapsed. There was no difference between dry and wet type recycled aggregate except for difference according to elapsed time. Waste official test method got the highest pH value between pH test methods. So unified pH test method need to test recycled aggregate pH.

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