Background[1-3]
Human platelet-type phosphofructokinase (PFKP) ELISA KIT is a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA). Standards with known concentrations of the substance to be tested and samples with unknown concentrations are added to the microwell enzyme plate for detection. First, the substance to be tested and the biotin-labeled antibody are incubated simultaneously. After washing, avidin-labeled HRP was added. After incubation and washing, unbound enzyme conjugates are removed, and then substrates A and B are added, and the enzyme conjugates are used simultaneously to produce color. The depth of the color is proportional to the concentration of the indicator in the sample.
Operation steps:
1. Mix all reagents thoroughly before use. Do not make the liquid produce a lot of foam to avoid adding a large number of bubbles when adding the sample, causing errors in the sample addition.
2. Determine the number of strips required based on the number of samples to be tested plus the number of standards. It is recommended that duplicate holes be made for each standard and blank hole. Each sample is determined according to its own quantity. If multiple holes can be used, try to make multiple holes.
3． Add 50ul of the diluted standard into the reaction well, and add 50ul of the sample to be tested into the reaction well. Immediately add 50ul of biotin-labeled antibody. Cover the membrane plate, shake gently to mix, and incubate at 37°C for 45 minutes.
4. Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 4 times. If washing with a plate washer, increase the number of washes by one.
5. Add 100ul of streptavidin-HRP to each well, shake gently to mix, and incubate at 37°C for 30 minutes.
6． Shake off the liquid in the wells, fill each well with washing liquid, shake for 30 seconds, shake off the washing liquid, and pat dry with absorbent paper. Repeat this 4 times. If washing with a plate washer, increase the number of washes by one.
7. Add 50ul of each of substrates A and B to each well, mix gently, and incubate at 37°C for 5 minutes. Avoid light.
8. Take out the enzyme plate and quickly add 50ul of stop solution. After adding the stop solution, measure the result immediately.
9. The OD value of each well was measured at a wavelength of 450 nm.
Apply[4][5]
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 reduced 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 production, and oxygen consumption.
7. Use Caki-1 cells stably transfected with sh PFKP to conduct tumor formation experiments in nude mice.
