Pyruvate Assay Kit

Catalog No. ABIN1000329

Product Details

Target: Pyruvate

Application: Biochemical Assay (BCA)

Characteristics: 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.

Components: Enzyme Mix: 10 mL. Dye Reagent: 120 µL. Standard: 400 µL 25 mM Pyruvate.

Material not included: Pipeting devices, centrifuge tubes, Clear flat-bottom 96-well plates, black 96-well or 384-well plates (e.g. Corning Costar) and plate reader.

Application Details

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

Protocol: 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 µMStandard Premix by mixing 10 µL of the 25 mM Standard and 490 µL H2O. Dilute Standard in distilled water. 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.

Calculation of Results:

Subtract blank OD (water, #8) from the standard OD values and plot the OD against standard concentrations.
Conversions: 1mM pyruvate equals 8.7 mg/dL or 87 ppm. FLUORIMETRIC

Restrictions: For Research Use only

Handling

Storage: -20 °C

References

Mukherjee, Wu, Barbour, Fang: "Lysophosphatidic acid activates lipogenic pathways and de novo lipid synthesis in ovarian cancer cells." in: The Journal of biological chemistry, Vol. 287, Issue 30, pp. 24990-5000, (2012) (PubMed).

Park, Baek, de Los Reyes, Yun, Hasenstein: "Transcriptome profiling characterizes phosphate deficiency effects on carbohydrate metabolism in rice leaves." in: Journal of plant physiology, Vol. 169, Issue 2, pp. 193-205, (2011) (PubMed).

Target Details

Target: Pyruvate

Background: Quantitative determination of pyruvate by colorimetric (570nm) or fluorimetric (530nm/590nm) methods.
Procedure: 20 min.

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. This 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 gamma em/ex = 585/530nm is directly proportional to pyruvate concentration in the sample.