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首页> 外文期刊>African Journal of Biotechnology >Preparation and characterization of the polyclonal antibody against GAR domain of microtubule actin cross-linking factor 1 (MACF1)
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Preparation and characterization of the polyclonal antibody against GAR domain of microtubule actin cross-linking factor 1 (MACF1)

机译：抗微管肌动蛋白交联因子1（MACF1）GAR域的多克隆抗体的制备和表征

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摘要

Growth arrest-specific 2 protein (Gas2) related domain (GAR domain), located at the C-terminal of microtubule actin cross-linking factor 1 (MACF1), plays a key role in microtubules (MTs) binding.?To prepare the polyclonal antibody against GAR domain, cDNA encoding 466 amino acids protein of GAR domain was amplified from MG-63 cell by RT-PCR. The amplified cDNA, that exhibited 99% identity to the published sequence, was cloned into prokaryotic expression vector pQE-80L for the expression of GAR domain. Polyclonal antibody was then developed by inoculation of New Zealand rabbit with the recombinant GAR protein (rGAR) (97% purity). After several doses of immunization, the titer of antiserum reached 1: 62500 when evaluated by ELISA. The polyclonal antibody was further confirmed by Western blot, which gave results of high specificity and sensitivity (1: 5000).?By using thepolyclonal antibody to?detect MACF1’s association with MTs, laser scanning confocal microscopy (LSCM) showed that widely expressed MACF1 was partially aligned along MT filaments in the cytoplasm and co-located with MTs.?This polyclonal antibody will be a valuable tool for the further studies?of MACF1 and its GAR domain.
机译：位于微管肌动蛋白交联因子1（MACF1）C端的生长停滞特异性2蛋白（Gas2）相关域（GAR域）在微管（MTs）结合中起关键作用。通过逆转录-聚合酶链反应（RT-PCR）从MG-63细胞中扩增了GAR结构域466个氨基酸蛋白的cDNA。与公开序列具有99％同一性的扩增cDNA被克隆到原核表达载体pQE-80L中以表达GAR结构域。然后通过用重组GAR蛋白（rGAR）（97％纯度）接种新西兰兔来开发多克隆抗体。数次免疫后，通过ELISA评估抗血清的效价达到1：62500。通过Western印迹进一步证实了该多克隆抗体，从而获得了高特异性和灵敏性的结果（1：5000）。通过使用多克隆抗体来检测MACF1与MT的关联，激光扫描共聚焦显微镜（LSCM）表明广泛表达的MACF1是该多克隆抗体将是进一步研究MACF1及其GAR结构域的有价值的工具。

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