Probe and method for detecting membrane of glucose transporter 4
A technology for glucose transport and probe, applied in biochemical equipment and methods, measurement devices, biological tests, etc., can solve the problems of difficult to achieve high-throughput screening of drugs, lack of high-throughput detection methods, and troublesome operation.
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[0036] We used two fluorescent proteins, pHluorin and TDimer2, to label amino acids 1–131 of the insulin-responsive aminopeptidase (IRAP) protein that fully co-localized with GLUT4 (exactly similar experimental results were also obtained using full-length IRAP, data not shown). We fused the pH-sensitive fluorescent protein pHluorin in front of IRAP131 (1-131 amino acids of the IRAP protein), and fused the red fluorescent protein TDimer2 insensitive to pH behind it to obtain the fusion protein TDimer2-IRAP-pHluorin. pHluorin does not emit light in an acidic environment, but emits green fluorescence when excited by 488nm light in a neutral or near-neutral environment. The constructed fluorescent protein is introduced into fat cells (lines) or muscle cells (lines) for expression through liposome or electroporation, and a stable cell line is established. Before stimulation by drugs such as insulin, pHluorin is in the acidic GLUT4 vesicles, so the cells only observe the red fluores...
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