Department of Cellular and Molecular Medicine, Laboratory of Peroxisome Biology and Intracellular Communication, KU Leuven, Leuven, Belgium.
Department of Biochemistry, Faculty of Pharmacy, Assiut University, Asyut, Egypt.
Methods Mol Biol. 2023;2643:183-197. doi: 10.1007/978-1-0716-3048-8_13.
The pyridine nucleotides NAD(H) and NADP(H) are key molecules in cellular metabolism, and measuring their levels and oxidation states with spatiotemporal precision is of great value in biomedical research. Traditional methods to assess the redox state of these metabolites are intrusive and prohibit live-cell quantifications. This obstacle was surpassed by the development of genetically encoded fluorescent biosensors enabling dynamic measurements with subcellular resolution in living cells. Here, we provide step-by-step protocols to monitor the intraperoxisomal NADPH levels and NAD/NADH redox state in cellulo by using targeted variants of iNAP1 and SoNar, respectively.
吡啶核苷酸 NAD(H) 和 NADP(H) 是细胞代谢中的关键分子，以时空精度测量它们的水平和氧化状态在生物医学研究中具有重要价值。传统方法评估这些代谢物的氧化还原状态具有侵入性，并且禁止对活细胞进行定量分析。通过开发遗传编码荧光生物传感器，克服了这一障碍，该传感器能够在活细胞中以亚细胞分辨率进行动态测量。在这里，我们提供了使用 iNAP1 和 SoNar 的靶向变体分别在细胞内监测过氧化物酶体内部 NADPH 水平和 NAD/NADH 氧化还原状态的逐步方案。
