α-Ketoglutarate (α-KG) is an important intermediate of the TCA cycle. It is used as an indicator of enhanced uptake of glucose metabolites into the TCA cycle and of enhanced glutaminolysis, the metabolic pathway for supplying α-ketoglutarate using glutamine as a substrate. α-Ketoglutarate plays an essential role in the production of glutamate and γ-aminobutyric acid (GABA) and contributes to the scavenging of reactive oxygen species. Additionally, a recent cancer study suggested that α-ketoglutarate behaves effectively for tumor suppression through the p53 protein1.
α-Ketoglutarate Assay Kit-Fluorometric enables the quantitation of α-ketoglutarate. This kit is optimized to quantify intracellular α-ketoglutarate by measuring the resorufin fluorescence generated by an enzymatic reaction (Ex：530 – 560 nm、Em：580 – 600 nm). This kit is formatted for 96-well microplates, and it is possible to measure multiple samples.
In the process from pretreatment of cultured cells to measurement using a plate reader, α-Ketoglutarate (α-KG) concentrations can be measured using the simple method required to add the reagent. The reproducibility of data, which is a common complaint from many customers who have used an existing product, was improved, and customers with no experience measuring α-KG concentrations can comfortably use this kit.
The two reasons that improve measurement reproducibility
① Sample pretreatment
In an existing product, pretreatment required cumbersome techniques, such as pH adjustment and filtration of a microscopic quantity, and this technical complexity contributed to data dispersion. On the other hand, using the simple method required to add the reagent, the α-Ketoglutarate Assay Kit-Fluorometric improves data dispersion caused during a pretreatment process.
② Measurement of α-Ketoglutarate
As with this kit, a plate assay for determining α-KG concentrations often uses the principle of measuring the amount of dye formed in the above reactions 1 and 2. In an existing product, data dispersion occurred when the two-step reaction proceeded simultaneously. However, the α-Ketoglutarate Assay Kit-Fluorometric uses the protocol that completes the two-step reaction, which successfully improved data dispersion arising from the chromogenic reaction.
Preparation of Standard Curve
α-Ketoglutarate levels in a sample can be measured by a calibration curve established with α-Ketoglutarate Standards included in this kit. If the α-Ketoglutarate levels are greater than or equal to 20 µmol/l, the sample must be diluted before measurement.
Change in intracellular metabolism of doxorubicin-treated A549 cells
Doxorubicin (DOX), which inhibits cell proliferation by acting on the G2/M phase of the cell cycle, is known to induce cellular senescence. A549 cells treated with DOX were compared to the control cells. As a result, intracellular α-KG concentrations increased in DOX-treated A549 cells. Additionally, the Cellular Senescence Detection Kit-SPiDER-β Gal was used to detect cellular senescence and the Cell Cycle Assay Solution Blue/Deep Red and the JC-1 MitoMP Detection Kit were used to confirm changes during the cell cycle and changes in mitochondrial membrane potential, respectively.
Q. How many samples can I measure?
A. The number of samples that can be recorded when the standard curve and sample are measured in triplicates.
(100 tests/kit: 12 samples) Please refer to the instruction manual for a 96-well plate layout.
Q. May I use color plates other than black plates for measurement?
A. You may not measure an accurate α-Ketoglutarate level if you use white or transparent plates; therefore, it is strongly recommended to use black plates.
Q. Our samples did not change in color, are there any reasons for this?
A. α-Ketoglutarate level contained in the sample may be lower than the detection limit (0.2 µmol/l) that can be measured using this kit. If you dilute the sample, the α-Ketoglutarate level contained in the diluted sample may be lower than 0.2 µmol/l. Please lower the dilution ratio and adjust the concentration of the sample over the detection limit that was measured using this kit.
Q. Can I store the working solution?
A. No, you cannot store the working solution. Please prepare this solution when you use it, as this solution becomes unstable when exposed to light. Please keep it in the dark after the preparation of the working solution. After the preparation, the working solution is stable in the dark at room temperature for approximately 2 hours.