Background[1-3]
Human phosphoenolpyruvate carboxykinase (PCK) ELISA KIT uses a double-antibody sandwich method to determine the level of human phosphoenolpyruvate carboxykinase (PCK) in specimens. Before collecting specimens, it is important to know whether the components to be tested are stable enough. Specimens that are tested on the same day after collection should be stored at 4°C for later use. If there are special reasons that require periodic collection of specimens, the specimens should be aliquoted in time and stored at -20°C or -70°C. Avoid repeated freezing and thawing. Specimens can be stored at 2-8℃ for 48 hours, and inorganic pigments can be stored at -20℃ for 1 month. Can be stored at -70 degrees for 6 months. Some hormone specimens require the addition of aprotinin. Use purified human phosphoenolpyruvate carboxykinase (PCK) capture antibody to coat the microwell plate to make a solid-phase antibody. Human phosphoenol pyruvate carboxykinase (PCK) is sequentially added to the coated microwells. , and then combined with the HRP-labeled detection antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB is added for color development. TMB is converted into blue under the catalysis of HRP enzyme and into the final yellow under the action of acid. The depth of the color is positively correlated with the human phosphoenolpyruvate carboxykinase (PCK) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm, and calculate the content of human phosphoenolpyruvate carboxykinase (PCK) in the sample through the standard curve.
Human phosphoenolpyruvate carboxykinase (PCK) is the main control point in regulating gluconeogenesis. Human phosphoenolpyruvate carboxykinase (PCK) together with GTP catalyzes the formation of phosphoenolpyruvate from oxaloacetate and releases carbon dioxide and GDP. Human phosphoenolpyruvate carboxykinase (PCK) expression can be regulated by insulin, glucocorticoids, glucagon, cAMP and diet.
Apply[4][5]
Human phosphoenolpyruvate carboxykinase (PCK) ELISA KIT is used to study the role of phosphoenolpyruvate carboxykinase in the metabolic process of tumor regeneration cells.
Using three-dimensional soft fibrin Matrigel (3D fibrin) separated and screened cancer stem cells (defined as tumor re-Huntsman dye-producing cells, TRCs) as a model to explore the cytoplasmic phosphorylation of one of the key enzymes of gluconeogenesis. Expression of enolpyruvate carboxykinase (PCK1) in melanoma regenerating cells and its metabolic function. Methods: (1) Use 3D fibrin to isolate TRCs in mouse melanoma B16 cells, liver cancer H22 cells, and lymphoma EL4 cells, and then use RT-PCR, Real time PCR and Western blot to detect these TRCs and corresponding control cells and mice Expression of PCK1 in undifferentiated mesenchymal stem cells (mMSCs) and embryonic stem cells (mESCs). (2) Use the gene expression data in the cancer gene database cbioportal to analyze the co-expression relationship between PCK1 and commonly used cancer stem cell markers such as CD133, ALDH1A1 and ABCG5 in multiple cells; and use Real time PCR to detect the expression of PCK1 in CD133+ and CD133-B16 cells. Expression differences among groups. (3) Immunohistochemical detection of PCK1 expression in 9 human melanoma clinical specimens; isolation of TRCs from fresh human primary melanoma specimens, and real-time PCR detection of PCK1 expression. (4) RT-PCR and Western blot detected the expression of the other two key enzymes FBP1 and G6Pase in gluconeogenesis in B16TRCs to explore whether PCK1 mediates the gluconeogenesis reaction. (5) After silencing the expression of PCK1 with siRNA, observe changes in the in vitro growth, glucose consumption, lactate release, and intermediate metabolites (such as serine/glycine, glycerol triphosphate) of B16 TRCs to explore the metabolic functions of PCK1. (6) Use siRNA to silence the expression of PCK1 in B16 or H22 TRCs and observe their tumor-forming ability in animals. (7) After silencing the expression of PCK1 in B16 TRCs with siRNA, relevant intermediate metabolites were supplemented to observe the impact on clone growth. (8) Observe the effect of PCK1 overexpression on the growth of B16 TRCs in vitro. (9) Construct B16 cells with EGFP fluorescence expression controlled by the PCK1 promoter, isolate the TRCs in the cells and place them in a normal hard environment for continuous culture, and use a fluorescence microscope to dynamically observe the fluorescence changes.
References
[1]Databases and Web Tools for Cancer Genomics Study[J].Yadong Yang,Xunong Dong,Bingbing Xie,Nan Ding,Juan Chen,Yongjun Li,Qian Zhang,Hongzhu Qu,Xiangdong Fang.Genomics,Proteomics&Bioinformatics.2015 (01)
[2]CD133:A cancer stem cells marker,is used in colorectal cancers[J].Fei Ren,Wei-Qi Sheng,Xiang Du.World Journal of Gastroenterology.2013(17)
[3]Dysregulated lipid metabolism in cancer[J].Feng Zhang,Guangwei Du,Department of Integrative Biology and Pharmacology,The University of Texas Health Science Center at Houston,Houston,TX 77030,United States.World Journal of Biological Chemistry.2012(08)
[4]ABCG5-positivity in tumor buds is an indicator of poor prognosis in node-negative colorectal cancer patients[J].lsabel Hostettler,Inti Zlobec,Luigi Terracciano,Alessandro Lugli.World Journal of Gastroenterology.2010(06)
[5]Li Yong. Research on the role of phosphoenolpyruvate carboxykinase in the metabolic process of tumor regeneration cells[D]. Huazhong University of Science and Technology, 2016.
