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: 10ml|Dye Reagent: 120ul|Standard: 400ul 25mM Pyruvate|Storage 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 100 500|2 80ul+20ul 100 400|3 60ul+40ul 100 300|4 40ul+60ul 100 200|5 30ul+70ul 100 150|6 20ul+80ul 100 100|7 10ul+90ul 100 50|8 0ul+100ul 100 0|Transfer 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–ODH2O|Slope|(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–FH2O|Slope|(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.