如何利用GST pulldown试剂盒进行蛋白相互作用检测? GST pulldown试剂盒是一种利用重组技术进行蛋白相互作用检测的方法。该试剂盒利用GST融合蛋白与目的蛋白的相互作用,通过GST与固相化在载体上的GTH亲和结合,从而实现蛋白的纯化和分离。 GST-pulldown方法主要包括三个步骤:首先,利用基因重组技术构建带有GST标签的原核表达载体;其次,通过原核表达系统表达带有GST标签的融合蛋白;最后,利用GST亲和纯化柱进行蛋白纯化,获得高纯度的融合蛋白,并进行蛋白间的相互作用检测。 该方法的基本原理是将目的蛋白与GST融合蛋白亲和固化在谷胱甘肽亲和树脂上,作为与目的蛋白亲和的支撑物。当目的蛋白溶液经过柱时,可以捕获与之相互作用的目的蛋白,然后通过SDS-PAGE电泳分析,证实两种蛋白间的相互作用或筛选出相应的目的蛋白。 为了获得可靠的实验结果,需要注意以下几点:首先,获得高纯度的GST融合蛋白可以减少实验的假阳性结果;其次,对融合蛋白进行质量控制,以确保蛋白的正确折叠;最后,在实验中加入核酸酶可以消除可能桥接到蛋白上的DNA和RNA,避免其对蛋白间相互作用的干扰。 GST pulldown试剂盒的应用 GST pulldown试剂盒可用于验证已知蛋白的相互作用,或筛选与已知蛋白相互作用的未知蛋白。例如,在青岛文昌鱼GST和FREP基因的研究中,利用GST pulldown试剂盒从青岛文昌鱼肠cDNA文库中分离出与脊椎动物肝脏功能相关的谷胱苷肽转硫酶(GST)和纤维蛋白原相关蛋白(FREP)基因,并对它们的结构、进化、表达和功能进行了研究。 参考文献 [1] Pathogen Recognition and Innate Immunity[J]. Shizuo Akira, Satoshi Uematsu, Osamu Takeuchi. Cell. 2006(4) [2] Presence and localization of antithrombin and its regulation after acute lipopolysaccharide exposure in amphioxus, with implications for the origin of vertebrate liver[J]. Yujun Liang, Shicui Zhang, Limin Lun, Li Han. Cell and Tissue Research. 2006(3) [3] Infectious non-self recognition in invertebrates: lessons from Drosophila and other insect models[J]. Julien Royet. Molecular Immunology. 2004(11) [4] The chordate amphioxus: an emerging model organism for developmental biology[J]. L.Z. Holland, V. Laudet, M. Schubert. Cellular and Molecular Life Sciences. 2004(18) [5] Cloning and phylogenetic analysis of an amphioxus myogenic bHLH gene AmphiMDF[J]. Jinduo Yuan, Shicui Zhang, Zhenhui Liu, Zhidong Luan, Gengxi Hu. Biochemical and Biophysical Research Communications. 2003(4) [6] Pattern recognition proteins in Manduca sexta plasma[J]. X.-Q. Yu, Y.-F. Zhu, C. Ma, J.A. Fabrick, M.R. Kanost. Insect Biochemistry and Molecular Biology. 2002(10) [7] The Role of Ficolins in Innate Immunity[J]. Misao Matsushita, Teizo Fujita. Immunobiology. 2002(4) [8] Ascidian and Amphioxus Adh Genes Correlate Functional and Molecular Features of the ADH Family Expansion During Vertebrate Evolution[J]. Cristian Ca?estro, Ricard Albalat, Lars Hjelmqvist, Laura Godoy, Hans J?rnvall, Roser Gonzàlez-Duarte. Journal of Molecular Evolution. 2002(1) [9] Bioinformatic tools for DNA/protein sequence analysis, functional assignment of genes and protein classification[J]. B. Rehm. Applied Microbiology and Biotechnology. 2001(5-6) [10] Parasite-responsive IgSF members in the snail Biomphalaria glabrata: characterization of novel genes with tandemly arranged IgSF domains and a fibrinogen domain[J]. Si-Ming Zhang, Pascale M.Léonard, Coen M. Adema, Eric S. Loker. Immunogenetics. 2001(8)查看更多