通过校准的免疫印迹法定量总 STAT3 和磷酸化 STAT3。
Quantification of total and phosphorylated STAT3 by calibrated western blotting.
机构信息
Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, 39106 Magdeburg, Germany.
Institute of Biology, Department of Systems Biology, Otto-von-Guericke University, 39106 Magdeburg, Germany; Center for Dynamic Systems: Systems Engineering (CDS), Otto-von-Guericke University, 39106 Magdeburg, Germany; Magdeburg Center for Systems Biology (MACS), Otto-von-Guericke University, 39106 Magdeburg, Germany; Center for Health and Medical Prevention (CHaMP), Otto-von-Guericke University, 39106 Magdeburg, Germany.
出版信息
STAR Protoc. 2023 Sep 15;4(3):102508. doi: 10.1016/j.xpro.2023.102508. Epub 2023 Sep 4.
Quantification of intracellular proteins is essential to understand signaling. Here, we describe quantification of the expression and phosphorylation of the transcription factor STAT3. We present isolation of total and phosphorylated STAT3 from cell lysates by immunoprecipitation, followed by SDS-PAGE and western blot together with known amounts of a calibrator protein that shares an epitope with the precipitated proteins. Finally, we explain how to relate the amount of precipitated protein to the amount of calibrator protein considering the efficiency of immunoprecipitation. For complete details on the use and execution of this protocol, please refer to Dittrich et al. (2012) and Reeh et al. (2019)..
定量检测细胞内蛋白对于理解信号转导至关重要。本文描述了转录因子 STAT3 的表达和磷酸化定量检测方法。我们通过免疫沉淀法从细胞裂解物中分离总 STAT3 和磷酸化 STAT3，然后进行 SDS-PAGE 和 Western blot，同时使用与沉淀蛋白具有相同表位的已知量校准蛋白。最后，我们解释了如何考虑免疫沉淀效率，将沉淀蛋白的量与校准蛋白的量相关联。有关此方案的使用和执行的完整详细信息，请参阅 Dittrich 等人（2012 年）和 Reeh 等人（2019 年）。
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