Encyclopedia of Experiments
Biological Techniques
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Encyclopedia of Experiments Biological Techniques
Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms

Pyruvate Assay for Estimation of Pyruvate Levels in Cellular Fractions of Worms

Transcription

Cellular levels of pyruvate — a key intermediate in energy-producing biochemical pathways — can be estimated using enzyme-based colorimetric assays.

To determine pyruvate levels in the cells of Caenorhabditis elegans — a non-parasitic nematode — take a cellular fraction prepared from adult worms containing pyruvate. Treat the solution, removing cellular proteins, and collect the clarified cellular fraction — the test sample — containing pyruvate. The absence of cellular proteins ensures an accurate determination of pyruvate levels.

Start by adding a working reagent containing the enzymes pyruvate oxidase and horseradish peroxidase, and a chromogenic dye. Incubate the mixture in the dark for an adequate duration.

During incubation, pyruvate oxidase reacts with pyruvate — its substrate — generating hydrogen peroxide as a byproduct. The absence of light prevents the spontaneous decomposition of hydrogen peroxide.

Horseradish peroxidase catalyzes the reaction between the hydrogen peroxide and the chromogenic dye, producing a colored product. The intensity of the colored reaction product is directly proportional to the pyruvate concentration in the sample.

Upon completion of the incubation period, place the sample inside a plate reader and measure the absorbance. Generate a standard curve using a serial dilution with known pyruvate concentrations.

Plot the measured absorbance value of the test sample on the curve to determine the pyruvate concentration in the test sample.

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