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  • Preparative free-solution isotachophoresis for separation of human plasma lipoproteins: apolipoprotein and lipid composition of HDL subfractions. 10828082

    We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (Böttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    ABS1030
    Produktbezeichnung:
    Anti-Apolipoprotein D Antibody

  • Immobilization of glutaryl-7-aminocephalosporanic acid acylase on silica gel and enhancement of its stability. 12665670

    Glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase is an enzyme that converts GL-7-ACA to 7-aminocephalosporanic acid, a starting material for semisynthetic cephalosporin antibiotics. In this study, optimal conditions for the immobilization of GL-7-ACA acylase were determined by experimental observations and statistical methods. The optimal conditions were as follows: 1.1 M phosphate buffer (pH 8.3) as buffer solution, immobilization temperature of 20 degrees C, and immobilization time of 120 min. Unreacted aldehyde groups were quenched by reaction with a low-molecular-weight material such as L-lysine, glycine, and ethanolamine after immobilization in order to enhance the activity of immobilized GL-7-ACA acylase. The activities of immobilized GL-7-ACA acylase obtained by using the low-molecular-weight materials were higher than those obtained by immobilized GL-7-ACA acylase not treated with low-molecular-weight materials. In particular, the highest activity of immobilized GL-7-ACA acylase was obtained using 0.4% (v/v) ethanolamine. We also investigated the effect of sodium cyanoborohydride in order to increase the stability of the linkage between the enzyme and the support. The effect on operational stability was obvious: the activity of immobilized GL-7-ACA acylase treated with 4% (w/w) sodium cyanoborohydride remained almost 100% after 20 times of reuse.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    03-104
    Produktbezeichnung:
    RIPAb+ CUGBP1 - RIP Validated Antibody and Primer Set

  • Modulation of choroidal neovascularization by subretinal injection of retinal pigment epithelium and polystyrene microbeads. 19158960

    The study was conducted to create a rapidly developing and reproducible animal model of subretinal choroidal neovascularization (CNV) that allows a time-dependent evaluation of growth dynamics, histopathologic features, and cytokine expression.C57BL/6 and chemoattractant leukocyte protein-2 deficient (DeltaCcl-2) mice were studied. Mice received single or combined subretinal injections of cultured retinal pigment epithelium (RPE; C57BL/6-derived), polystyrene microbeads, or phosphate buffer solution (PBS). Fluorescence angiograms were performed over a period of 3 weeks. Mice were euthanized on post inoculation day 3, 7, 10, 14, or 21, and their eyes were evaluated by light, confocal, and electron microscopy.CNV membranes occurred in all study groups with an overall incidence of 94.3%. They extended in the subretinal space through central breaks in Bruch's membrane. CNV lesions were characterized by dynamic changes such as initiation, active inflammatory, and involution stages. CNV thickness peaked around PI day 7 and was greater in mice that received combined injections of RPE and microbeads or RPE cells alone. Small lesions developed in the control groups (microbeads or PBS only), in DeltaCcl-2, and old C57BL/6 mice. Variable expression of cytokines and growth factors was detected within the membranes.Our murine model represents a reliable approach inducing CNV growth by subretinal injection of either RPE cells alone or RPE cells and microbeads. The development of CNV lesions is a dynamic process that relies in part on macrophage trafficking and age.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere
    Produktbezeichnung:
    Mehrere

  • Nanostructured polymer matrix for oligonucleotide separation 16892480

    A nanostructured copolymer matrix has successfully separated oligonucleotides with high resolution by CE using a very short separation channel which simulates the real microchip condition for the first time. The triblock copolymer, E45B14E45 (B20-5000) with E, B, and subscript denoting oxyethylene, oxybutylene, and segment length, respectively, has a unique temperature-dependent viscosity-adjustable property and a dynamic coating ability in 16TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA in Milli-Q water). The B-block is hydrophilic at low temperatures, e.g., 47C, and the polymer solution has a very low viscosity of about 100 cP in a 32.5% w/v solution. At room temperatures, the B-block becomes hydrophobic due to a breakdown of hydrogen bonds between B-blocks and water, and the polymer matrix forms a body-centered cubic structure at high concentrations. Oligonucleotide sizing markers ranging from 8 to 32 base could be successfully separated with one-base resolution in a 1.5 cm long separation channel by E45B14E45 in its gel-like state.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    Mehrere