Colorimetric Assay for Quantification of Lactate in Test Samples

In living cells, under anaerobic conditions, the enzyme lactate dehydrogenase converts pyruvate — an end product of glycolysis — to lactate — a key metabolite. The amount of lactate produced in the cells reflects their metabolic status.

To quantify the intracellular lactate concentration, begin with a test sample of protein-free, nematode cell lysate. Supplement other wells of a multi-well plate with a lactate solution of various known concentrations.

The lysate lacks all proteinaceous enzymes, including endogenous lactate dehydrogenase, ensuring accurate lactate quantification.

Add a lactate assay buffer into each well to maintain the appropriate pH for optimum enzymatic activity. Supplement the wells with a reaction mix containing the lactate dehydrogenase enzyme, nicotinamide-adenine dinucleotide — NAD — a coenzyme, and a colorimetric probe and incubate.

During incubation, lactate dehydrogenase catalyzes the oxidation of cellular lactate to pyruvate, with a simultaneous reduction of NAD into NADH. The generated NADH reacts with the colorimetric probe to produce a purple product.

Spectroscopically measure the purple solution's absorbance for the test sample and lactate standards