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.
Simple, 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.
Key Features:Sensitive 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 Fluorometric Assay:s
Simple 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.
Applications:Direct Assays: pyruvate in biological samples
Drug Discovery/Pharmacology: effects of drugs on pyruvate metabolism.
Kit Contents:Enzyme Mix: 10mlDye Reagent: 120ulStandard: 400ul 25mM PyruvateStorage conditions. The kit is shipped on dry ice. Store all reagents at -20°C. Shelf life of three months after receipt
Precautions: 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.
Colorimetric Procedure:Note: SH-group containing reagents (e.g. mercaptoethanol, DTT) may interfere with this assay and should be avoided in sample preparation
1. 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
No Premix+H2O Vol (ul) Pyruvate (uM)1 100ul+0ul 100 5002 80ul+20ul 100 4003 60ul+40ul 100 3004 40ul+60ul 100 2005 30ul+70ul 100 1506 20ul+80ul 100 1007 10ul+90ul 100 508 0ul+100ul 100 0Transfer 10ul standards and 10ul samples into separate wells of a clear flat-bottom 96-well plate
2. 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
3. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm)
Note: 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.
Calculation:Subtract 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[Pyruvate] =ODSAMPLE–ODH2OSlope(uM)ODSAMPLE and ODH2O are optical density values of the sample and water
Conversions: 1mM pyruvate equals 8.7 mg/dL or 87 ppm.
Fluorometric Procedure:For Fluorometric Assays, the linear detection range is 0.2 to 50uM pyruvate. Dilute the Standards prepared in Colorimetric Procedure: 1:10 in H2O
Transfer 10ul standards and 10ul samples into separate wells of a black 96-well plate
Add 90ul Working Reagent (see Colorimetric Procedure:). Tap plate to mix
Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm
If assays in 384-well plate are desired, use 5ul Standards and 45ul Working Reagent. The pyruvate concentration of Sample is calculated as[Pyruvate] =FSAMPLE–FH2OSlope(uM)
Materials Required, But Not Provided:Pipeting devices, centrifuge tubes, Clear flat-bottom 96-well plates, black 96-well or 384-well plates (e.g. Corning Costar) and plate reader.
