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. BioAssay Systems' 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 10 μL samples. Linear detection range in 96-well plate: 2 to 500 μM (17 μg/dL to 4.4 mg/dL) pyruvate for colorimetric assays and 0.2 to 50 μM for fluorimetric assays.

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: 10 mL

Dye Reagent: 120 μL

Standard: 400 μL 25 mM 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 500 μM Standard Premix by mixing 10 μL of the 25 mM Standard and 490 μL H2O. Dilute Standard in distilled water as follows.

Transfer 10 μL standards and 10 μL samples into separate wells of a clear flat-bottom 96-well plate.

2. For each reaction well, mix 94 μL Enzyme Mix and 1 μL Dye Reagent in a clean tube. Transfer 90 μL 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 500 μM, dilute sample in water and repeat the assay. Multiply result by the dilution factor.

ODSAMPLE and ODH2O are optical density values of the sample and water.

Conversions: 1mM pyruvate equals 8.7 mg/dL or 87 ppm.

For fluorimetric assays, the linear detection range is 0.2 to 50 μM pyruvate. Dilute the Standards prepared in Colorimetric Procedure 1:10 in H2O. Transfer 10 μL standards and 10 μL samples into separate wells of a black 96-well plate. Add 90 μL 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 5μL Standards and 45 μL Working Reagent. The pyruvate concentration of Sample is calculated as