• Title, Summary, Keyword: affinity label

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Affinity labeling of the Vacuolar Arginine Transporter in Neurospora crassa (Neurospora crassa의 액포에 존재하는 arginine transporter의 표지방법)

  • ;Weiss, R. L.
    • Korean Journal of Microbiology
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    • v.27 no.2
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    • pp.108-116
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    • 1989
  • Based on the specificty of recognition of the vacuolar arginine transporter, N-p-nitrobenzoxycarbonyl (NBZ)-L-arginyl diazomethane was synthesized and used as an affinity label specific for the arginine transporter. This arginyl derivative ingibited both ATP-dependent and independent L-arginine transport into vacuolar membrane vesicles. When vacuolar proteins were labeled with radioactive NBZ arginyl diazomethane, the binding was irreversible, detached by treatment with base and blocked by treatment with cysteinyl blocking groups suggesting cysteine as a labeling site.

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Bioelectrocatalyzed Signal Amplification for Affinity Interactions at Chemically Modified Electrodes

  • Hyun C. Yoon;Kim, Hak-Sung
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.9 no.2
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    • pp.107-111
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    • 2004
  • A comparative study was performed to evaluate the signal amplification strategies in electrochemical affinity sensing, which included the direct electron transfer and diffusible-group mediated electron transfer between label enzymes that were specifically bound to target proteins and chemically modified electrode surfaces. As a platform surface for affinity recognition reactions, a double functionalized poly(amidoamine) dendrimer monolayer that was modified with ferrocene and biotin groups was constructed on a gold surface. With the chemically modified electrode, a model affinity sensing with avidin was investigated. The advantages of adopting the diffusible-group mediated signaling strategy were demonstrated in terms of signal sensitivity and stability.

Label-free Detection of Biomolecular Specific Interaction by Optical Biosensors (광 바이오센서를 이용한 비표지 생계물질들의 특이 상호작용력의 측정)

  • 김의락;최정우
    • KSBB Journal
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    • v.17 no.1
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    • pp.1-13
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    • 2002
  • Label-free optical methods for the monitoring of interactions between biological molecules have become increasingly popular within the last decade. A rising number of publications have demonstrated the benefits of direct biomolecular interaction analysis(BIA) for biology and biochemistry, such as antigen-antibody Interactions, receptor-ligand interactions, protein-DNA, DNA- intercalator, and DNA-DNA interactions. This article gives an overview of the historical development, principle and application of label-free optical biosensor to examine the functional characteristics of biospecific interaction, such as kinetics, affinity, and binding position of biomolecular between an immobilized species at the transducer surface and its dissolved binding partner.

Affinity Labeling of E. coli GTP Cyclohydrolase I by a Dialdehyde Derivative of Guanosine Triphosphate

  • Ahn, Chi-Young;Park, Sang-Ick;Kim, Ju-Myeong;Yim, Jeong-Bin
    • BMB Reports
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    • v.28 no.1
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    • pp.72-78
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    • 1995
  • Time-dependent inactivation of E. coli GTP cyclohydrolase I with a 2',3'-dialdehyde derivative of GTP (oGTP) was directed to the active site of the enzyme, and was dependent on the concentration of oGTP. The kinetics of inactivation were biphasic with a rapid reaction occurring immediately upon exposure of the enzyme to oGTP followed by a slow rate of inactivation. The $K_i$ value of oGTP for the enzyme was 0.25 mM. Inactivation was prevented by preincubation of the enzyme with GTP, the substrate of the enzyme. At 100% inactivation, 2.3 mol of [8.5'-$^3H$]oGTP were bound per each enzyme subunit, which consists of two identical polypeptides. The active site residue which reacted with the affinity label was lysine. oGTP interacted selectively with the ${\varepsilon}$-amino group of lysine in the GTP-binding site to form a morpholine-like structure which was stable without sodium borohydride treatment. However, triphosphate group was eliminated during the hydrolysis step. To identify the active site of the enzyme, [8.5'-$^3H$]oGTP-labeled enzyme was cleaved by endoproteinase Lys-C, and the $^3H$-labeled peptide was purified by HPLC. The amino acid sequence of the active site peptide was Pro-Ser-Leu-Ser-Lys, which corresponds to the aminoterminal sequence of the enzyme.

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Label-free Detection of the Transcription Initiation Factor Assembly and Specific Inhibition by Aptamers

  • Ren, Shuo;Jiang, Yuanyuan;Yoon, Hye Rim;Hong, Sun Woo;Shin, Donghyuk;Lee, Sangho;Lee, Dong-Ki;Jin, Moonsoo M.;Min, Irene M.;Kim, Soyoun
    • Bulletin of the Korean Chemical Society
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    • v.35 no.5
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    • pp.1279-1284
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    • 2014
  • The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

Interaction of Cytochrome c and Cytochrome c Oxidase Studied by Spin-Label EPR and Site-Directed Mutagenesis

  • Park, Hee-Young;Chun, Sun-Bum;Han, Sang-Hwa;Lee, Kwang-Soon;Kim, Kyung-Hoon
    • BMB Reports
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    • v.30 no.6
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    • pp.397-402
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    • 1997
  • A thiol-specific spin label was attached to cysteine-102 of yeast cytochrome c and electron paramagnetic resonance (EPR) spectra were measured as a function of added cytochrome c oxidase concentration. The intensity decreased due to line broadening as cytochrome c formed a complex with cytochrome c oxidase and reached a minimum when the ratio of cytochrome c to cytochrome c oxidase became one. Replacement of either Lys-72 or Lys-87 of cytochrome c by Glu did not result in a significant change in binding affinity. Interestingly the K72E mutant, unlike K87E, had a much lower rate of electron transfer than the wild type. These results indicate that many positively charged residues as a group participate in complex formation but Lys-72 might be important for cytochrome c to be locked in an orientation for an efficient electron transfer. A stoichiometry of 1 was also confirmed by optical absorption of the cytochrome c-cytochrome c oxidase complex which had been run through a gel chromatography cloumn to remove unbound cytochrome c. The EPR spectrum of this 1:1 complex, however, was a mixture of two components. This explains a biphasic kinetics for a single binding site on cytochrome c oxidase without invoking conformational transition.

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Purification of the Vacuolar Arginine Transporter from Neurospora crassa (Neurospora crassa로부터 arginine transporter의 순수분리)

  • ;Weiss, R. L.
    • Korean Journal of Microbiology
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    • v.27 no.2
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    • pp.117-123
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    • 1989
  • Radioactive N-$\alpha$-p-nitrobenzoxycarbonyl (NBZ)-L-[2,$3-^{3}$H] arginyl diazomethane was used as an affinity label for the vacuolar arginine transporter in Neurospora crassa. Vacuolar matrix proteins were removed by fracturing the membranes with freeze-thaw method in dry ice/ethanol bath. Vacuolar membrane proteins were then wasged with 500mM NaCl to remove ionically bound derivatives and peripheral membrane proteins from vacuolar membranes. After dissolved in 1% Titon X-100, dissolved vacuolar memvrane proteins were separated with molecular sieve column chromatography, anion and cation exchange chromatographies. The arginine transporter was purified giving the purification factor of 1136.

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Immunoassay of haptoglobin and transferrin with proteinG-containing QCM sensor chip and unpurified antiserum (Protein G를 포함하는 수정미소저울 센서 칩과 정제되지 않은 항혈청을 이용한 헵토글로빈과 트랜스페린의 면역분석)

  • Ha, In-Young;Choi, Suk-Jung
    • Journal of Sensor Science and Technology
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    • v.17 no.5
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    • pp.380-386
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    • 2008
  • Quartz crystal microbalance immunosensor has a capacity to perform a label-free and real time detection of a trace amount of analyte through the specific interaction between antibody and antigen. However, immobilization of antibody molecules on the sensor surface is a troublesome procedure for researchers who are not experienced in chemistry. Protein G has a specific affinity to antibody and would serve as a capturing agent for antibody when immobilized on the sensor surface. In this work, we prepared a protein G sensor chip by immobilizing protein G on the surface of quartz crystal microbalance and examined its capability to detect human haptoglobin or human transferrin with unpurified corresponding antiserum. Specific and dose dependent response was observed when the protein G chip was used for detection of antigens after saturated with antiserum. We also verified several advantageous aspects of the protein G chip such as improved flexibility and sensitivity.

Develop ECO-FREE high concentration Full black dye using transfer printing and application technology (전사날염용 ECO-FREE 고농도 Full Black 염료개발과 응용기술)

  • Cho, Ho-Hyun;Chung, Myung-Hee;Lee, A-Ram
    • Journal of the Korea Fashion and Costume Design Association
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    • v.19 no.2
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    • pp.39-48
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    • 2017
  • Transfer printing is a method to combine printing and dyeing technology by the use of sublimation. It is an environmentally-friendly printing method that saves costs, reduces the production processes by the omission of the washing process, and saves time by maintaining quality. Due to the development of transfer printing, a high value added printing technology is available now but color fastness to sublimation of the printing products is still low since there are few dyes that have an affinity to the fabrics and the application technology is still inadequate. Specially, in case of high concentration black dyes, eco-label type black dyes, which is a substitution for general dispersal dyes, have been developed while general dispersal black dyes are still used, creating issues such as color differences on the surface and back side of the fabrics and contamination by friction after transfer printing. There are also some restricted substances such as allergens. To address these issues, high concentration black dyes and application technology that are environmentally-friendly and that have over 16 K/S through the use of single dyes with excellent color fastness, fixation ability, and similar melting temperature were developed for this study.

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A Multi-Layer Graphical Model for Constrained Spectral Segmentation

  • Kim, Tae Hoon;Lee, Kyoung Mu;Lee, Sang Uk
    • Proceedings of the Korean Society of Broadcast Engineers Conference
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    • pp.437-438
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    • 2011
  • Spectral segmentation is a major trend in image segmentation. Specially, constrained spectral segmentation, inspired by the user-given inputs, remains its challenging task. Since it makes use of the spectrum of the affinity matrix of a given image, its overall quality depends mainly on how to design the graphical model. In this work, we propose a sparse, multi-layer graphical model, where the pixels and the over-segmented regions are the graph nodes. Here, the graph affinities are computed by using the must-link and cannot-link constraints as well as the likelihoods that each node has a specific label. They are then used to simultaneously cluster all pixels and regions into visually coherent groups across all layers in a single multi-layer framework of Normalized Cuts. Although we incorporate only the adjacent connections in the multi-layer graph, the foreground object can be efficiently extracted in the spectral framework. The experimental results demonstrate the relevance of our algorithm as compared to existing popular algorithms.

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