Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity
Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity
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DOI：
10.1016/j.ab.2004.02.012
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发表时间：
2004-06-15
影响因子：
2.9
通讯作者：
Palecek, SP
中科院分区：
文献类型：
--
作者：
Brueggemeier, SB;Kron, SJ;Palecek, SP
我们报道了一种基于聚丙烯酰胺的蛋白质固定化策略的发展和表征，用于表面结合蛋白的分析，包括浓度检测、结合亲和力和酶动力学。将谷胱甘肽-S-转移酶(GST)融合蛋白标记在丙烯酸基上，并通过与丙烯酸单体的共聚反应将其附着在丙烯酸功能化的玻璃表面。GST-绿色荧光蛋白融合蛋白的特异性粘附量是非丙烯酸标记的GST-GFP非特异性粘附量的7倍以上，直接测量绿色荧光蛋白的荧光强度可检测到0.32 ng/mm(2)的GST-GFP，而间接抗体检测法的检测下限降至0.080 ng/mm(2)。基于聚丙烯酰胺的表面附着策略也被用来测量底物被激酶c-Src磷酸化的动力学。在溶液中反应的米氏动力学常数为K-m=2.7+/-1.0um，V-max=8.1+/-3.1(任意单位)。表面固定化底物催化反应的动力学参数分别为K-m=0.36+/-0.033µm和V-max=9.7+/-0.63，与共聚物中丙烯酰胺浓度无关。这种表面附着策略应该适用于蛋白质组学领域，并解决与蛋白质微阵列开发相关的变性和脱水问题。(C)2004年，爱思唯尔公司出版。
We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) 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. Michaelis-Menten kinetic constants for the reaction occurring in solution were K-m = 2.7 +/- 1.0 muM and V-max = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K-m = 0.36 +/- 0.033 muM and V-max = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development. (C) 2004 Published by Elsevier Inc.