Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Esam S.B. Salem; Kazutoshi Murakami; Toshimasa Takahashi; Elise Bernhard; Vishnupriya Borra; Mridula Bethi; Takahisa Nakamura

doi:10.3791/58323

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

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Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

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08:04 min

October 23, 2018

DOI: 10.3791/58323-v

Esam S.B. Salem*^1,2, Kazutoshi Murakami*², Toshimasa Takahashi², Elise Bernhard², Vishnupriya Borra², Mridula Bethi², Takahisa Nakamura^2,3,4

¹Department of Pharmacology and Systems Physiology, College of Medicine,University of Cincinnati, ²Division of Endocrinology,Cincinnati Children's Hospital Medical Center, ³Division of Developmental Biology,Cincinnati Children's Hospital Medical Center, ⁴Department of Pediatrics, College of Medicine,University of Cincinnati

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在这里, 我们提出了一个分离健康和功能的原发性小鼠肝细胞的协议。通过非放射性标记基底检测肝新生蛋白合成的说明, 帮助了解肝脏能量代谢稳态背景下蛋白质合成的机理。

该方法有助于回答各种代谢疾病研究领域有关肥胖、非酒精性脂肪肝和二型糖尿病中肝能和蛋白质生物合成分子变化的关键问题。该技术的主要优点是，它便于通过非放射性标签基板分离功能性原位小鼠肝细胞和检测肝新生蛋白。这种方法的视觉演示是至关重要的，因为灌注步骤是很难学习，因为小和薄的小鼠血管。

对于肝脏灌注，小心插入24表导管到劣质维纳卡瓦或 IVC 只是在分叉与正确的肾静脉。拆下导管针，保持 IVC 内导管的位置，并使用连接器将 24 表导管与灌注管连接。开始用温暖的HSS减去四毫升的永久流速来循环肝脏，迅速切断静脉，排出内部血液。

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