Colorimetric Assay for Quantification of Lactate in Test Samples
Transcrição
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 at 570 nanometers. Plot the absorbance values of the lactate standards and generate the calibration curve.
Finally, compare the absorbance value of the sample with the lactate standards to determine the amount of lactate present in the test sample.
