Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling
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Author:
Date:
2017-06-05
[Abstract] Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the background fluorescence for non-specific attachments. In this protocol, rabbit antibodies were raised against the synthetic peptide of CyDiv protein (Mandakovic et al., 2016). The secondary antibody conjugated to the fluorophore Alexa488 was used due to its different emission range in comparison to the autofluorescence of the cyanobacterium.
[摘要] 诸如免疫荧光的技术被广泛用于确定蛋白质的亚细胞分布。在这里我们报告一种免疫定位蛋白质的方法。 PCC7120通过荧光显微镜检测荧光团结合的抗体。该方法改善了蓝细菌细胞的透化性,并使非特异性附着物的背景荧光最小化。在该方案中,针对CyDiv蛋白质的合成肽(Mandakovic等人,2016)产生兔抗体。使用与荧光团Alexa488缀合的二抗,因为与蓝细菌的自发荧光相比,其发射范围不同。
背景 蓝细菌的免疫荧光已广泛用于细胞鉴定和计数研究(Jin等人,2016)。然而,在蓝细菌中蛋白质的免疫定位尚未有效地实现。定位蛋白质最复发的方法是通过将感兴趣的蛋白质融合到具有不同发射波长的荧光蛋白(绿色荧光蛋白)(与蓝细菌自发荧光相比))以及随后使用落射荧光或共聚焦显微镜(Flores < em,et="" al。,2016;=""> &NBSP;蓝细菌细胞的结构性质是应用免疫荧光技术的主要挑战。它们由内膜(IM),肽聚糖层(PG)和外膜(OM)组成(Rippka,1988; Baulina,2012; Jin等人,2016),附加外多糖层(鞘)。鞘细胞均存在于单细胞和丝状蓝细菌中(Kehr and Dittmann,2015),其厚度,组成和外观取决于生长条件,代谢状态,细胞分化及其他外部和内部参数(Jin et al。 ...
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Single Molecule RNA FISH in Arabidopsis Root Cells
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Author:
Date:
2017-04-20
[Abstract] Methods that allow the study of gene expression regulation are continually advancing. Here, we present an in situ hybridization protocol capable of detecting individual mRNA molecules in plant root cells, thus permitting the accurate quantification and localization of mRNA within fixed samples (Duncan et al., 2016; Rosa et al., 2016). This single molecule RNA fluorescence in situ hybridization (smFISH) uses multiple single-labelled oligonucleotide probes to bind target RNAs and generate diffraction-limited signals that can be detected using a wide-field fluorescence microscope. We adapted a recent version of this method that uses 48 fluorescently labeled DNA oligonucleotides (20 mers) to hybridize to different portions of each transcript (Raj et al ...
[摘要] 允许研究基因表达调控的方法不断前进。在这里,我们提出了一种能够检测植物根细胞中单个mRNA分子的原位杂交方案,从而允许mRNA在固定样品内的准确定量和定位(Duncan等人, ,2016; Rosa等人,2016)。这种单分子RNA荧光原位杂交(smFISH)使用多个单标记寡核苷酸探针结合靶RNA并产生可以使用宽场荧光显微镜检测的衍射限制信号。我们调整了该方法的最新版本,该方法使用48个荧光标记的DNA寡核苷酸(20个)与每个转录物的不同部分杂交(Raj等人,2008)。这种方法简单易行,具有很好的应用于任何遗传背景的优点。
虽然已经开发了单分子FISH来定量测量培养细胞,组织切片和整体无脊椎动物生物体的单细胞水平的mRNA,但该方法未被优化用于植物中的单细胞。由于植物组织的内源性自发荧光,植物中的荧光成像是相当有挑战性的。在这里,我们报告一种检测植物中单RNA分子的方法。我们描述在固定的拟南芥根瘤胃细胞内的单一转录物的检测和自动计数。该方法产生隔离的单元和单细胞层,其与红色和远红色染料一起使用可以最大化限制背景噪声的信噪比。
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Indirect Immunofluorescence Assay in Chlamydomonas reinhardtii
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Author:
Date:
2016-07-05
[Abstract] Determining the protein localization is essential to elucidate its in vivo function. Fluorescence-tagged proteins are widely used for it, but it is sometimes difficult to express tagged proteins in Chlamydomonas. Alternatively, indirect immunofluorescence assay is also one of the widely used methods and many reports determining the localization of Chlamydomonas proteins using this method are published. Here, we introduce a protocol of indirect immunofluorescence assay adapted from our papers reporting LCIB (CO2-recycling factor in the vicinity of pyrenoid; Yamano et al., 2010), LCI1 (plasma membrane-localized inorganic carbon transporter; Ohnishi et al., 2010), HLA3 (plasma membrane-localized ABC-type bicarbonate transporter; Yamano et ...
[摘要] 确定蛋白质定位是阐明其体内功能所必需的。荧光标记的蛋白质被广泛使用,但有时难以在衣原体中表达标记的蛋白质。或者,间接免疫荧光测定也是广泛使用的方法之一,并且公开了使用该方法确定衣藻蛋白定位的许多报道。在这里,我们介绍间接免疫荧光测定方案改编自我们的论文报告LCIB(在芘类化合物附近的CO 2 - 再循环因子; Yamano等人,2010), LCI1(质膜定位的无机碳转运蛋白; Ohnishi等人,2010),HLA3(浆膜定位的ABC-型碳酸氢盐转运蛋白; Yamano等人,2015) )和LCIA(叶绿体包膜阴离子通道; Yamano等人,2015)在莱茵衣藻(Chlamydomonas reinhardtii)中。这里描述的协议可以用于观察其他藻类细胞感兴趣的蛋白质。
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