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    Hydrogels for biomolecule analysis and corresponding method to analyze biomolecules
    1.
    发明授权
    Hydrogels for biomolecule analysis and corresponding method to analyze biomolecules 有权
    用于生物分子分析的水凝胶和分析生物分子的相应方法

    公开(公告)号：US07588906B2

    公开(公告)日：2009-09-15

    申请号：US11305671

    申请日：2005-12-16

    申请人： Shawn B. Brueggemeier ,  Stephen J. Kron ,  Sean P. Palecek ,  Laurie Parker ,  Stephen Brian Henry Kent

    发明人： Shawn B. Brueggemeier ,  Stephen J. Kron ,  Sean P. Palecek ,  Laurie Parker ,  Stephen Brian Henry Kent

    IPC分类号： C12Q1/37 ,  G01N33/53

    CPC分类号： G01N33/6842 ,  G01N33/6851 ,  H01J49/04

    摘要： Polyacrylamide-based methods of fabricating surface-bound peptide and protein arrays, the arrays themselves, and a method of using the arrays to detect biomolecules and to measure their concentration, binding affinity, and kinetics are described. Peptides, proteins, fusion proteins, protein complexes, nucleic acids, and the like, are labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through a copolymerization with acrylic monomer. The specific attachment of glutathione S-transferase-green fluorescent protein (GST-GFP) fusion protein was more than 7-fold greater than the nonspecific attachment of non-acrylic labeled GST-GFP. Surface-attached GST-GFP (0.32 ng/mm2) was detectable by direct measurement of green fluorescent protein fluorescence and this lower detection limit was reduced to 0.080 ng/mm2 using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src which is encoded by the Rous Sarcoma virus. The surface attachment strategy is applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.

    摘要翻译： 描述了基于聚丙烯酰胺的制备表面结合肽和蛋白质阵列的方法，阵列本身以及使用阵列检测生物分子并测量其浓度，结合亲和力和动力学的方法。 肽，蛋白质，融合蛋白，蛋白质复合物，核酸等用丙烯酸部分标记，并通过与丙烯酸类单体的共聚连接到丙烯酸官能化的玻璃表面上。 谷胱甘肽S-转移酶绿色荧光蛋白（GST-GFP）融合蛋白的特异性附着比非丙烯酸标记的GST-GFP的非特异性附着大7倍以上。 通过直接测量绿色荧光蛋白荧光检测表面附着的GST-GFP（0.32ng / mm2），并使用间接抗体检测将该检测下限降低至0.080ng / mm2。 基于聚丙烯酰胺的表面附着策略也用于通过由Rous肉瘤病毒编码的激酶c-Src来测量底物磷酸化的动力学。 表面附着策略适用于蛋白质组学领域，涉及与蛋白质微阵列开发相关的变性和脱水问题。