Generation and Characterization of a Rat Monoclonal Antibody Specific for Multiple Red Fluorescent Proteins
Generation and Characterization of a Rat Monoclonal Antibody Specific for Multiple Red Fluorescent Proteins
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DOI：
10.1089/hyb.2008.0031
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发表时间：
2008-10-01
期刊：
影响因子：
--
通讯作者：
Cardoso, M. Cristina
中科院分区：
文献类型：
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作者：
Rottach, Andrea;Kremmer, Elisabeth;Cardoso, M. Cristina
荧光蛋白（FP）被广泛用作体内报告分子，并且可以在几乎整个可见光谱的多种颜色中获得。FP基因融合到任何蛋白质靶点，为研究蛋白质定位和动力学提供了强大的工具。在从水母Aequorea victoria中分离出典型的绿色荧光蛋白（GFP）后，在珊瑚Discosoma sp.中发现了红色荧光蛋白（DsRed），其提供了与细胞自发荧光更好的光谱分离，并允许融合蛋白的荧光跟踪。然而，DsRed的专性四聚化对其在活细胞成像中的应用造成了严重的问题。红色祖细胞的后续诱变产生了几种单体红色FP（mRFP1、mCherry、mOrange、mPlum等）。这些改进的红色FP的特征在于更高的亮度和光稳定性，完整的发色团成熟，并承诺用于生物成像和荧光标记的各种功能。在这里，我们报告的第一个大鼠单克隆抗体，（单克隆抗体）的产生和表征多种红色FP，指定为多红5F8。我们证明了Multi-red 5178是一种对DsRed衍生物和相应融合蛋白具有高亲和力和特异性的单克隆抗体，并且它适用于ELISA、免疫印迹、免疫沉淀和免疫荧光测定。应用我们的多功能抗体，同一个红色荧光蛋白标签不仅可用于显微镜研究，还可用于多种生化分析。
Fluorescent proteins (FP) are widely used as in vivo reporter molecules and are available in Multiple colors spanning almost the entire visible light spectrum. Genetically fused to any protein target, FPs offer a powerful tool to study protein localization and dynamics. After the isolation of the prototypical green fluorescent protein (GFP) from the jellyfish Aequorea victoria, a red fluorescent protein (DsRed) was discovered in the coral Discosoma sp. that provided a better spectral separation from cellular autofluorescence and allowed multicolor tracking of fusion proteins. However, the obligate tetramerization of DsRed caused serious problems for its use in live-cell imaging. Subsequent mutageneses of the red progenitor have resulted in several monomeric red FPs (mRFP1, mCherry, mOrange, mPlum, etc). These improved red FPs are characterized by higher brightness and photostability, complete chromophore maturation, and promise a wide variety of features for biological imaging and multicolor labeling. Here we report the generation and characterization of the first rat monoclonal antibody, (MAb) against multiple red FPs, designated as multi-red 5F8. We demonstrate that multi-red 5178 is a MAb with high affinity and specificity against the DsRed derivatives and corresponding fusion proteins, and that it is Suitable for ELISA, immunoblotting, immunoprecipitation, and immunofluorescence assays. Applying Our versatile antibody, one and the same red fluorescent protein tag can be used to perform not only microscopic Studies, but also multiple biochemical assays.