A panel of monoclonal antibodies to cytoplasmic GW bodies and the mRNA binding protein GW182
A panel of monoclonal antibodies to cytoplasmic GW bodies and the mRNA binding protein GW182
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DOI：
10.1089/153685903321947996
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发表时间：
2003-04-01
期刊：
影响因子：
--
通讯作者：
Fritzler, MJ
中科院分区：
文献类型：
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作者：
Eystathioy, T;Chan, EKL;Fritzler, MJ
GW182是一种mRNA结合蛋白，以60个重复的甘氨酸(G)：色氨酸(W)基序为特征，定位于被称为GW小体（gwb）的细胞质结构中。目前的证据表明，这种独特的蛋白质在mRNA加工中起作用。为了更详细地研究GW182和gwb在细胞和组织中的作用，包括它们在mRNA加工中的作用，我们开发了四种结合人重组GW182蛋白的单克隆抗体（mab）。这些单克隆抗体可用于培养细胞和组织的Western blot分析和间接免疫荧光（IIF）。特别值得注意的是，其中一种单克隆抗体2D6可以通过抗原回收法（ARM）鉴定福尔马林固定组织和石蜡包埋组织中的GW182和gwb。本研究中描述的所有单克隆抗体都能免疫沉淀GW182蛋白。使用重叠的15-mer肽进行表位定位，代表全长GW182，表明这些单克隆抗体的主要抗体结合域是不同的。这些单克隆抗体是细胞生物学家和病理学家研究新型GW182蛋白在组织培养细胞以及冷冻保存或存档组织中的位置和功能的宝贵工具。
GW182 is a mRNA binding protein characterized by 60 repeats of glycine (G):tryptophan (W) motifs and is localized in cytoplasmic structures referred to as GW bodies (GWBs). Current evidence suggests that this unique protein plays a role in mRNA processing. To enable a more detailed study of GW182 and GWBs in cells and tissues, including their role in mRNA processing, we developed four monoclonal antibodies (MAbs) that bind the human recombinant GW182 protein. These MAbs can be used for Western blot analysis and indirect immunofluorescence (IIF) on cultured cells and tissues. Of special interest, one of the MAbs, 2D6, can be used to identify GW182 and GWBs in formalin-fixed and paraffin-embedded tissues after using an antigen retrieval method (ARM). All the MAbs described in this study immunoprecipitate the GW182 protein. Epitope mapping using overlapping 15-mer peptides representing the full-length GW182 showed that the major antibody-binding domains of these MAbs are distinct. These MAbs are valuable tools for cell biologists and pathologists to study the location and function of the novel GW182 protein in tissue culture cells, as well as cryopreserved or archived tissues.