Background[1-3]
Human platelet-type phosphofructokinase (PFKP) ELISA kit uses a double-antibody one-step sandwich method enzyme-linked immunosorbent assay (ELISA). To the microwells pre-coated with human platelet-type phosphofructokinase (PFKP) capture antibody, add specimen, standard, and HRP-labeled detection antibody in sequence, incubate and wash thoroughly. The substrate TMB is used for color development. TMB is converted into blue under the catalysis of peroxidase, and converted into the final yellow under the action of acid. The depth of the color is positively correlated with the human platelet-type phosphofructokinase (PFKP) in the sample. Use a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm and calculate the sample concentration.
Operation steps
1. Dilute the antibody to 1-10ug/ml with buffer. Add 0.1ml to the reaction well and keep overnight at 4°C. The next day, discard the solution in the wells and wash three times with washing buffer.
2. Add sample: Add 0.05ml of a certain dilution of the sample to be tested into the coated reaction well, and incubate at 37°C for 1 hour. Then wash (make blank holes, negative control holes and positive control holes at the same time).
3. Add enzyme-labeled antibody: Add 0.05ml of freshly diluted enzyme-labeled antibody (dilution after titration) to each reaction well. Incubate at 37°C for 0.5 to 1 hour and wash.
4. Add substrate solution for color development: Add 0.1 ml of temporarily prepared TMB substrate solution to each reaction well, and incubate at 37°C for 10 to 30 minutes.
5. Terminate the reaction: add 0.05ml of 2M sulfuric acid to each reaction well
6. Result judgment: The results can be observed directly with the naked eye on a white background: the darker the color in the reaction well, the stronger the positive degree. The negative reaction is colorless or extremely light, depending on the depth of the color.
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Research on platelet isoform phosphofructokinase promoting metabolic reprogramming and cell proliferation in clear cell renal cell carcinoma
Metabolic changes in clear cell renal cell carcinoma (cc RCC) include aerobic glycolysis, increased pentose phosphate pathway activity and decreased oxidative phosphorylation. Phosphofructokinase (PFK), a key enzyme in the glycolysis pathway, has L, M and P subtypes and is distributed in different tissues. So far it is still unclear which isoform of PFK is abundantly expressed in cc RCC, whether it plays an important role in promoting aerobic glycolysis and macromolecule biosynthesis, and supporting cell proliferation.
Research methods 1.q PCR and Western blot were used to detect the expression of PFKP, PFKM and PFKL in clear cell renal cell carcinoma tissues. Immunohistochemical detection of PFKP protein levels in clear cell renal cell carcinoma tissues.
2. q PCR and Western blot were used to detect the expression of PFKP in normal renal tubular epithelial cells HK-2, renal clear cell carcinoma cell lines Caki-1, 769-p and 786-O cells.
3. si RNA interferes with the expression of PFKP in Caki-1, 769-p and 786-O cells, and detects cell proliferation, cell cycle and apoptosis.
4. si RNA interferes with the expression of PFKP in Caki-1, 769-p and 786-O cells, and detects cellular glucose uptake, lactate production, oxygen consumption and PFK1 enzyme activity.
5. Establish Caki-1 cells stably transfected with sh PFKP, and use LCMS to detect changes in the metabolome.
6. Establish Caki-1 cells stably transfected with shp53, and explore the role of p53 in inhibiting PFKP-induced changes in cell proliferation, cell cycle, apoptosis, glucose uptake, lactate formation and oxygen consumption.
7. Use Caki-1 cells stably transfected with sh PFKP to conduct tumorigenesis experiments in nude mice.
Research results 1. The platelet isoform of phosphofructokinase (PFKP) is the main PFK isoform in human renal clear cell carcinoma and is significantly up-regulated in tumors.
2. Inhibiting PFKP not only reduces the proliferation of renal clear cell carcinoma cells by inducing cell cycle arrest and apoptosis, but inhibiting PFKP can also lead to a decrease in glycolysis, pentose phosphate pathway, and nucleotide synthesis, accompanied by tricarboxylic acid synthesis. Acid cycle activation.
3. Activation of the p53 gene is one of the mechanisms responsible for the slowed proliferation and metabolic abnormalities of renal clear cell cancer cells after knocking down the PF original dispersant KP imported from Germany.
