Pyruvate Assay Kit, BioAssay(TM)

Pyruvate is a key intermediate in cellular metabolic pathways. Pyruvate can be converted to carbohydrates via gluconeogenesis, to fatty acids or energy through acetyl-CoA, to the amino acid alanine and to ethanol. Abnormal levels of pyruvate have been linked to liver diseases and metabolic disorders.\n\nSimple, direct and automation-ready procedures for measuring pyruvate concentrations find wide applications in research and drug discovery. Pyruvate assay uses a single Working Reagent that combines pyruvate oxidase and hydrogen peroxide determination in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to pyruvate concentration in the sample.\n\nKey Features:\nSensitive and accurate. Use as little as 10ul samples. Linear detection range in 96-well plate: 2 to 500uM (17 ug/dL to 4.4 mg/dL) pyruvate for Colorimetric Assay:s and 0.2 to 50uM for Fluorimetric Assay:s.\nSimple and convenient. The procedure involves addition of a single working reagent and incubation for 30 min at room temperature, compatible for HTS assays. Improved reagent stability. The optimized formulation has greatly enhanced the reagent and signal stability.\n\nApplications:\nDirect Assays: pyruvate in biological samples.\nDrug Discovery/Pharmacology: effects of drugs on pyruvate metabolism.\n\nKit Contents:\nEnzyme Mix: 10ml\nDye Reagent: 120ul\nStandard: 400ul 25mM Pyruvate\nStorage conditions. The kit is shipped on dry ice. Store all reagents at -20 degrees C. Shelf life of three months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nColorimetric Procedure:\nNote: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation.\n1. Equilibrate all components to room temperature. Prepare a 500uM Standard Premix by mixing 10ul of the 25mM Standard and 490ul H2O. Dilute Standard in distilled water as follows.\nNo Premix+H2O Vol (ul) Pyruvate (uM)\n1 100ul+0ul 100 500\n2 80ul+20ul 100 400\n3 60ul+40ul 100 300\n4 40ul+60ul 100 200\n5 30ul+70ul 100 150\n6 20ul+80ul 100 100\n7 10ul+90ul 100 50\n8 0ul+100ul 100 0\nTransfer 10ul standards and 10ul samples into separate wells of a clear flat-bottom 96-well plate.\n2. For each reaction well, mix 94ul Enzyme Mix and 1ul Dye Reagent in a clean tube. Transfer 90ul Working Reagent into each assay well. Tap plate to mix. Freeze unused reagents for future use.\n3. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm).\nNote: if the Sample OD is higher than the Standard OD at 500uM, dilute sample in water and repeat the assay. Multiply result by the dilution factor.\n\nCalculation:\nSubtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations. Determine the slope using linear regression fitting. The pyruvate concentration of Sample is calculated as\n[Pyruvate] =\nODSAMPLE

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SPECIFICATIONS

Catalog Number

P9543-85

Size

1Kit

References

1. Hansen JL, Freier EF. (1978). Direct assays of lactate, pyruvate, beta-hydroxybutyrate, and acetoacetate with a centrifugal analyzer. Clin Chem. 24(3): 475-9.\n2. Sutherland DV, Barns AM, Ross CA. (1995). Trypanosoma evansi: measurement of pyruvate production as an indicator of the drug sensitivity of isolates in vitro. Trop Med Parasitol. 46(2): 93-8.\n3. Chariot P. et al (1994). Optimal handling of blood samples for routine measurement of lactate and pyruvate. Arch Pathol Lab Med. 118(7): 695-7.