• Title/Summary/Keyword: 2-DE gel

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Purification and Characterization of an Extracellular $\beta$-Glucosidase from Monascus purpureus

  • Daroit, Daniel J.;Simonetti, Aline;Hertz, Plinho F.;Brandelli, Adriano
    • Journal of Microbiology and Biotechnology
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    • v.18 no.5
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    • pp.933-941
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    • 2008
  • An extracellular $\beta$-glucosidase produced by Monascus purpureus NRRL1992 in submerged cultivation was purified by acetone precipitation, gel filtration, and hydrophobic interaction chromatography, resulting in a purification factor of 92-fold. A $2^2$ central-composite design (CCD) was performed to find the best temperature and pH conditions for enzyme activity. Maximum activity was observed in a wide range of temperature and pH values, with optimal conditions set at $50^{\circ}C$ and pH 5.5. The $\beta$-glucosidase showed moderate thermostability, was inhibited by $HgCl_2$, $K_2Cr_O_4$, and $K_2Cr_2O_7$, whereas other reagents including $\beta$-mercaptoethanol, SDS, and EDTA showed no effect. Activity was slightly stimulated by low concentrations of ethanol and methanol. Hydrolysis of p-nitrophenyl-$\beta$-D-glucopyranoside (pNPG), cellobiose, salicin, n-octyl-$\beta$-D-glucopyranoside, and maltose indicates that the $\beta$-glucosidase has broad substrate specificity. Apparently, glucosyl residues were removed from the nonreducing end of p-nitrophenyl-$\beta$-D-cellobiose. $\beta$-Glucosidase affinity and hydrolytic efficiency were higher for pNPG, followed by maltose and cellobiose. Glucose and cellobiose competitively inhibited pNPG hydrolysis.

Quantitative Proteomics Towards Understanding Life and Environment

  • Choi, Jong-Soon;Chung, Keun-Yook;Woo, Sun-Hee
    • Korean Journal of Environmental Agriculture
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    • v.25 no.4
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    • pp.371-381
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    • 2006
  • New proteomic techniques have been pioneered extensively in recent years, enabling the high-throughput and systematic analyses of cellular proteins in combination with bioinformatic tools. Furthermore, the development of such novel proteomic techniques facilitates the elucidation of the functions of proteins under stress or disease conditions, resulting in the discovery of biomarkers for responses to environmental stimuli. The ultimate objective of proteomics is targeted toward the entire proteome of life, subcellular localization biochemical activities, and the regulation thereof. Comprehensive analysis strategies of proteomics can be classified into three categories: (i) protein separation via 2-dimensional gel electrophoresis (2-DE) or liquid chromatography (LC), (ii) protein identification via either Edman sequencing or mass spectrometry (MS), and (iii) proteome quantitation. Currently, MS-based proteomics techniques have shifted from qualitative proteome analysis via 2-DE or 2D-LC coupled with off-line matrix assisted laser desorption ionization (MALDI) and on-line electrospray ionization (ESI) MS, respectively, toward quantitative proteome analysis. In vitro quantitative proteomic techniques include differential gel electrophoresis with fluorescence dyes. protein-labeling tagging with isotope-coded affinity tags, and peptide-labeling tagging with isobaric tags for relative and absolute quantitation. In addition, stable isotope-labeled amino acids can be in vivo labeled into live culture cells via metabolic incorporation. MS-based proteomics techniques extend to the detection of the phosphopeptide mapping of biologically crucial proteins, which ale associated with post-translational modification. These complementary proteomic techniques contribute to our current understanding of the manner in which life responds to differing environment.

Development of Methods for Protein Extraction from Three Major Korean Fermented Soy Foods for 2-Dimensional Gel and Mass Spectrometric Analyses

  • Lim, Jin-Kyu
    • Journal of Applied Biological Chemistry
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    • v.51 no.3
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    • pp.88-94
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    • 2008
  • Three different protein extraction methods-phenol extraction, trichloroacetic acid (TCA) precipitation, and desalting/TCA precipitation-were compared to determine the optimal reproducible high resolution 2-dimensional (2-D) electrophoresis for each chungkugjang, doenjang, and kochujang samples. The soluble proteins from Chungkugjang extracted by phenol were separated with high reproducibility and resolution, and gained 1.75- to 3-fold more protein spots on 2-D gel than those from the other methods. On the contrary, the extracted proteins from doenjang and kochujang treated by desalting/TCA precipitation method showed about 1.5- to 3.3-fold more protein spots on 2-D gel. Using the established methods, the changes in the protein profiles of the fermented soy foods were monitored during the fermentation period by 2-DE. One of the major proteins in soy, $\beta$-conglycinin $\alpha$-subuint, and some proteins with unknown functions were localized on 2-D gel as the protease-resistant proteins throughout the fermentation period of doenjang. Changes in the protein profile monitored by the established methods can provide basic information on unfolding the mechanisms of the generation of biofunctional activity in the fermented soy foods.

Discovery-Driven Exploration Method in Lung Cancer 2-DE Gel Images Using the Data Cube (데이터 큐브를 이용한 폐암 2-DE 젤 이미지에서의 예외 탐사)

  • Shim, Jung-Eun;Lee, Won-Suk
    • The KIPS Transactions:PartD
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    • v.15D no.5
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    • pp.681-690
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    • 2008
  • In proteomics research, the identification of differentially expressed proteins observed under specific conditions is one of key issues. There are several ways to detect the change of a specific protein's expression level such as statistical analysis and graphical visualization. However, it is quiet difficult to handle the spot information of an individual protein manually by these methods, because there are a considerable number of proteins in a tissue sample. In this paper, using database and data mining techniques, the application plan of OLAP data cube and Discovery-driven exploration is proposed. By using data cubes, it is possible to analyze the relationship between proteins and relevant clinical information as well as analyzing the differentially expressed proteins by disease. We propose the measure and exception indicators which are suitable to analyzing protein expression level changes are proposed. In addition, we proposed the reducing method of calculating InExp in Discovery-driven exploration. We also evaluate the utility and effectiveness of the data cube and Discovery-driven exploration in the lung cancer 2-DE gel image.

Electric properties of $ Ta_2$$O_5$ thin films by sol-gel method (졸-겔법에 의한$ Ta_2$$O_5$ 박막의 전기적 특성)

  • 유영각;이준웅
    • Electrical & Electronic Materials
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    • v.10 no.1
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    • pp.61-67
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    • 1997
  • We have studied dielectric properties of sol-gel derived tantalum oxide thin films as the insulators. As the sample is annealed from 300.deg. C to 700.deg. C, it is found amorphous below 600.deg. C and crystalline over it. Dielectric constant is maximum(18.6) when Ta$_{2}$O$_{5}$ film was annealed at 400.deg. C. It is found that dielectric strength in Ta$_{2}$O$_{5}$ film annealed at 400.deg. C (1.5MV/cm) increases and then decreases over annealed at 500.deg. C. This phenomenon was attributed to pinhole effect and crystallization. The de conduction properties can be interpreted by Poole-Frenkel effect.ect.

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Characterization of ZnO/TiO2 Nanocomposites Prepared via the Sol-Gel Method

  • Hellen, Nalumaga;Park, Hyun;Kim, Kyung-Nam
    • Journal of the Korean Ceramic Society
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    • v.55 no.2
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    • pp.140-144
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    • 2018
  • $ZnO/TiO_2$ nanocomposites were synthesized via a modified sol-gel technique by incorporating 30 and 70 wt% $TiO_2$ nanopowder into a ZnO sol-gel matrix. Zinc acetate dihydrate was used as the ZnO precursor and de-ionized water as the solvent, while titanium oxysulfate was employed for the synthesis of $TiO_2$ nanopowder. The synthesized $ZnO/TiO_2$ nanocomposites were characterized by x-ray diffraction, UV-vis spectroscopy, scanning electron microscopy, and transmission electron microscopy. The $ZnO/TiO_2$ nanocomposites showed both the ZnO (wurtzite) and $TiO_2$ (anatase) phases. The average ZnO crystallite size of the $ZnO/TiO_2$ nanocomposites was found to be about 26.3 nm. The TEM results confirmed that spherical $TiO_2$ particles were embedded in the ZnO matrix. $TiO_2$ particles attached onto the rod-like ZnO particles were also observed. The $ZnO/TiO_2$ nanocomposites exhibited optical absorption properties superior to those of pure ZnO and $TiO_2$.

Opto-Electrical Study of Sol-Gel Derived Antimony Doped Tin Oxide Films on Glass

  • De, Arijit
    • Transactions on Electrical and Electronic Materials
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    • v.16 no.1
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    • pp.5-9
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    • 2015
  • Optical and electrical properties were studied for Antimony doped tin oxide thin films from precursors containing 10, 30, 50, and 70 atom% of Sb deposited on bare sodalime silica, barrier layer coated sodalime silica, and pure silica glass substrates by sol-gel spinning technique. The direct band gaps were found to vary from 3.13~4.12 eV when measured in the hv range of 2.5~5.0 eV, and varied from 4.22~5.08 eV when measured in the range of 4.0~7.0 eV. Indirect band gap values were in the range of 2.35~3.11 eV. Blue shift of band gap with respect to bulk band gap and Moss-Burstein shift were observed. Physical thickness of the films decreased with the increase in % Sb. Resistivity of the films deposited on SLS substrate was in the order of $10^{-2}$ ohm cm. Sheet resistance of the films deposited on barrier layer coated soda lime silica glass substrate was found to be relatively less.

The Analysis of Seminal Plasma Proteins by Two-Dimensional Polyacrylamide Gel Electrophoresis (2-DE) in Hanwoo (Korean Native Cattle)

  • Lee, Yong-Seung;Song, Eun-Ji;Yoo, Han-Jun;Park, Joung-Jun;Cheong, Hee-Tae;Yang, Boo-Keun;Park, Choon-Keun
    • Journal of Embryo Transfer
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    • v.25 no.4
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    • pp.281-286
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    • 2010
  • This study was to evaluate the protein profile of seminal plasma using 2-DE in Hanwoo. Seminal plasma was harvested from five mature Hanwoo, and seminal plasma protein was extracted by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $300\;{\mu}l$. Immobilized pH gradient (IPG) strip was used 18 cm and 3~11 NL. SDS-PAGE was used 12% acrylamide gel. Each gels were visualized by comassie brilliant blue and silver staining. These spots were analyzed by MALDI-TOF MS and searched on NCBInr. The result, 20 proteins of 36 protein spots were searched through peptide sequencing on the NCBInr. 8 proteins profiled by 2-DE were proved through previous bovine studies and the name of each protein was albumin, nucleobindin, clusterin, TIMP-2, spermadhesin Z13, spermadhesin-1 and BSP proteins (BSP 30 kDa and BSP A1/A2). 12 new proteins were ATP synthase, protein MAK16 homolog, Transmembrane protein 214, E3 ubiquitin-protein ligase BRE1A, dual serine/threonine and tyrosine protein kinase, tissue factor pathway inhibitor 2, alpha-actinin-4, RUN domain-containing protein 3B, catenin alpha-1, protein-glutamine gamma-glutamyltransferase 2, plakophilin-1 and inter-alpha-trypsin inhibitor heavy chain H1 has not been previously described in the bovine seminal plasma study. These proteins may be contribute to define the type of proteins affecting fertility of male and improve the fertilizing ability of semen in Hanwoo.

Automated Method of Landmark Extraction for Protein 2DE Images based on Multi-dimensional Clustering (다차원 클러스터링 기반의 단백질 2DE 이미지에서의 자동화된 기준점 추출 방법)

  • Shim, Jung-Eun;Lee, Won-Suk
    • The KIPS Transactions:PartD
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    • v.12D no.5 s.101
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    • pp.719-728
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    • 2005
  • 2-dimensional electrophoresis(2DE) is a separation technique to identify proteins contained in a sample. However, the image is very sensitive to its experimental conditions as well as the quality of scanning. In order to adjust the possible variation of spots in a particular image, a user should manually annotate landmark spots on each gel image to analyze the spots of different images together. However, this operation is an error-prone and tedious job. This thesis develops an automated method of extracting the landmark spots of an image based on landmark profile. The landmark profile is created by clustering the previously identified landmarks of sample images of the same type. The profile contains the various properties of clusters identified for each landmark. When the landmarks of a new image need to be fount all the candidate spots of each landmark are first identified by examining the properties of its clusters. Subsequently, all the landmark spots of the new image are collectively found by the well-known optimization algorithm $A^*$. The performance of this method is illustrated by various experiments on real 2DE images of mouse's brain-tissues.

The effects of blocking the oxygen in the air during the polymerization of sealant (광중합 시 공기 중 산소의 차단이 치면열구전색제의 중합에 미치는 영향)

  • Oh, You-Hyang;Lee, Nan-Young;Lee, Sang-Ho
    • Journal of the korean academy of Pediatric Dentistry
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    • v.33 no.3
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    • pp.365-376
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    • 2006
  • The purpose of this study was to evaluate the efficacy of blocking the oxygen in the air during the polymerization of sealant. All curing were performed with various light curing units under the application of oxygen gel barrier, stream of nitrogen and carbon dioxide gas for inhibition of oxygen diffusion into sealant surface. The results of present study can be summarized as follows : 1. The amount of eluted TEGDMA form the specimens cured with all the three different light units in the stream of $N_2$ and $CO_2$ gas and application of Oxygen gel barrier($DeOx^{(R)}$) were significantly lower than in the room-air atmosphere (Control) (p<0.05). 2. In the $DeOx^{(R)}$ application, the amount of eluted TEGDMA the specimen cured with PAC light for 10seconds was less than that cured in the stream of $N_2$ and $CO_2$ atmospheric conditions (p<0.05) 3. In the LED using 10 or 20sec irradiation times under the stream of $N_2$ and $CO_2$, the eluted TEGDMA showed to be no statistically significant difference (p>0.05). 4. The microhardness from the specimens cured with all the three different light units under each treated conditions were significantly higher than in the room-air atmosphere (p<0.05). 5. The surface treatment by $DeOx^{(R)}$, $N_2$ and $CO_2$ reduces the thickness of oxygen inhibited layer by sp proximately 49% of the untreated control value.

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