Microcalorimetry of α - ketoglutarate dehydrogenase activity detection kit

α -Ketoglutarate dehydrogenase（α-KGDH）Instructions for Activity Detection Kit
Micro method
Attention: The reagents of this product have been changed. Please pay attention to and strictly follow the instructions in this manual.
Item Number：BC0715
specification：100T/96S
Product composition: Before use, please carefully check whether the volume of the reagent matches the volume inside the bottle. If you have any questions, please contact the staff of Solaibao in a timely manner.

Preparation of solution:

1. Reagent 2: It is a volatile reagent. Seal it as soon as possible after use and store it at -20 ℃;

2. Reagent 5: Take one vial of reagent 5 before use and add it1ML reagent three, fully dissolved, unused reagent2-8℃save4week

3. Reagent 6: Take one vial of Reagent 6 before use and add it1.5ML reagent three, fully dissolved, unused reagent-20 ℃ packaging and storage4Zhou, avoid repeated freeze-thaw cycles;

4. Reagent 7: Take one vial of Reagent 7 before use and add it1ML reagent three, fully dissolved, unused reagent-20 ℃ packaging and storage4Zhou, avoid repeated freeze-thaw cycles (can be done after dissolving 1 powder)100TIn order to extend the usage time, this product is given more1Powder powder);

5. Reagent 8: Take one vial of Reagent 8 before use and add it0.4mLDistilled water, fully dissolved, unused reagents-20℃Packaging and preservation4Zhou, avoid repeated freeze-thaw cycles;

6. Preparation of working fluid: Take 8.05 in sequence before useML reagent 30.2 mLReagent 4, 1mLReagent 5, 1.25mLReagent 6, 0.5mLReagent Seven(In total)11mL, about55T）Mix thoroughly for later use and prepare as needed.

Product Description:
α-KGDH（EC 1.2.4.2）Widely present in the mitochondria of animals, plant microorganisms, and cultured cells, it is one of the key enzymes regulating the tricarboxylic acid cycle, catalyzingα -Oxidative decarboxylation of ketoglutaric acid to produce succinyl CoA*A.
α-KGDHcatalysisα -Ketoglutaric acidNAD⁺Generate succinyl auxiliary with auxiliary * A*ACarbon dioxide andNADH，NADHin340nmThere are characteristic absorption peaks, toNADHThe generation rate representationα-KGDHActivity.

Attention: It is recommended to select 2-3 samples with significant expected differences for pre experiments before the experiment. If the absorbance value of the sample is not within the measurement range, it is recommended to dilute or increase the sample size for testing.
Equipment and supplies to be provided:
UV spectrophotometer/ELISA reader, water bath/Constant temperature incubator, desktop centrifuge, adjustable pipette, trace quartz colorimetric dish/96KongUVBoard, mortar/Homogenizer, ice, and distilled water.
Operation steps:For specific operation steps, please refer to the attachment of "Product Information"

1. During the measurement process, all reagents and samples are placed on ice to prevent denaturation and inactivation.

2. The temperature of the reaction solution in the colorimetric dish must be maintained at 37 ℃ or25℃Take a small beaker and fill it with a certain amount of37℃or25℃Distilled water, put this beaker into it37℃or25℃In a water bath. Place the colorimetric dish along with the reaction solution in this beaker during the reaction process.

3. It is best for two people to conduct this experiment simultaneously, one for color comparison and the other for timing, to ensure the accuracy of the experimental results.

4. Measuring tubeΔAValue in0.01-0.25Between, if measuring the tubeΔAValue greater than0.25The sample needs to be diluted.

5. Due to the presence of a certain concentration of protein (approximately 1mg/mL) in the extraction solution, the protein content of the extraction solution itself needs to be subtracted when determining the protein concentration of the sample.

Experimental Example:

1. Take 0.1g of barnyard grass for sample processingDilute the supernatant by 2 timesFollow the measurement steps and use a trace quartz colorimetric dish to measurecalculateΔAdetermination=A4-A3=0.3243-0.3115=0.0128，ΔAblank=A2-A1=0，Calculate enzyme activity based on sample quality:

α-KGDHactivity(U/g mass)=1488.5×(ΔAdetermination-ΔAblank)÷W×2(Dilution ratio)=381.056 U/gquality

1. Take 0.1g of mouse liver for sample processing,4 ℃ 11000g centrifugation10minAfter taking the supernatantFollow the measurement steps and use a trace quartz colorimetric dish to measurecalculateΔAdetermination=A4-A3=1.2123-0.9623=0.2500，ΔAblank=A2-A1=0，Calculate enzyme activity based on sample quality:

α-KGDHactivity(U/g mass)=1488.5×(ΔAdetermination-ΔAblank)÷W=3721.25 U/gquality
Related publications:

1. Jianyun Yue,Changjian Du,Jing Ji,et al. Inhibition of α-ketoglutarate dehydrogenase activity afects adventitious root growth in poplar via changes in GABA shunt. Planta. July 2018; (IF3.06)

2. Xiao Li,Qi Zhao,Jianni Qi,et al. lncRNA Ftx promotes aerobic glycolysis and tumor progression through the PPARγ pathway in hepatocellular carcinoma. International Journal of Oncology. May 2018; (IF3.571)

References:
Park L C H, Calingasan N Y, Sheu K F R, et al. Quantitative α-ketoglutarate dehydrogenase activity staining in brain sections and in cultured cells[J]. Analytical biochemistry, 2000, 277(1): 86-93.
Related product series:
BC2150/BC2155 Citric acid (CA) content detection kit
BC0950/BC0955 Succinate dehydrogenase (SDH) activity detection kit
BC0380/BC0385 Pyruvate dehydrogenase (PDH) activity detection kit
BC2160/BC2165 Mitochondrial Isocitrate Dehydrogenase (ICDHm) Activity Detection Reagent

Microcalorimetry of α - ketoglutarate dehydrogenase activity detection kit Microcalorimetry of α - ketoglutarate dehydrogenase activity detection kit