John Laura, Drescher Malte
Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Konstanz, Germany.
Bio Protoc. 2018 Apr 5;8(7):e2798. doi: 10.21769/BioProtoc.2798.
One of the most exciting perspectives for studying bio-macromolecules comes from the emerging field of in-cell spectroscopy, which enables to determine the structure and dynamics of bio-macromolecules in the cell. In-cell electron paramagnetic resonance (EPR) spectroscopy in combination with micro-injection of bio-macromolecules into oocytes is ideally suited for this purpose. oocytes are a commonly used eukaryotic cell model in different fields of biology, such as cell- and development-biology. For in-cell EPR, the bio-macromolecules of interest are microinjected into the oocytes upon site-directed spin labeling. The sample solution is filled into a thin glass capillary by means of Nanoliter Injector and after that microinjected into the dark animal part of the oocytes by puncturing the membrane cautiously. Afterwards, three or five microinjected oocytes, depending on the kind of the final in-cell EPR experiment, are loaded into a Q-band EPR sample tube followed by optional shock-freezing (for experiment in frozen solution) and measurement (either at cryogenic or physiological temperatures) after the desired incubation time. The incubation time is limited due to cytotoxic effects of the microinjected samples and the stability of the paramagnetic spin label in the reducing cellular environment. Both aspects are quantified by monitoring cell morphology and reduction kinetics.
研究生物大分子最令人兴奋的前景之一来自细胞内光谱学这一新兴领域，它能够确定细胞内生物大分子的结构和动力学。细胞内电子顺磁共振（EPR）光谱结合将生物大分子显微注射到卵母细胞中，非常适合这一目的。卵母细胞是生物学不同领域中常用的真核细胞模型，如细胞生物学和发育生物学。对于细胞内EPR，在进行定点自旋标记后，将感兴趣的生物大分子显微注射到卵母细胞中。通过纳升注射器将样品溶液装入细玻璃毛细管中，然后小心地刺穿细胞膜，将其显微注射到卵母细胞的暗动物部分。之后，根据最终细胞内EPR实验的类型，将三到五个显微注射的卵母细胞装入Q波段EPR样品管中，在所需的孵育时间后，进行可选的速冻（用于冷冻溶液实验）和测量（在低温或生理温度下）。由于显微注射样品的细胞毒性作用和顺磁性自旋标记在还原性细胞环境中的稳定性，孵育时间受到限制。通过监测细胞形态和还原动力学对这两个方面进行量化。
