Pyruvate Assay Kit, BioAssay™

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.

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.

Direct Assays: pyruvate in biological samples.
Drug Discovery/Pharmacology: effects of drugs on pyruvate metabolism.

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.

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.

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.

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)

Pipeting devices, centrifuge tubes, Clear flat-bottom 96-well plates, black 96-well or 384-well plates (e.g. Corning Costar) and plate reader.

This product as supplied is intended for research use only, not for use in human, therapeutic or diagnostic applications without the expressed written authorization of United States Biological.