Gen5 version required: 1.0 or higher
Basis for the Assay:
Lactic acid is a by-product of carbohydrate metabolism. Lactate is converted to pyruvate by the enzyme lactate dehydrogenase along with the deduction of NAD to NADH. NADH absorbs light at 340 nm, while NAD does not. Therefore this reaction can be followed by the increase in absorbance at 340 nm. Under conditions where NAD and Lactate dehydrogenase concentrations are in excess of lactate, the formation of NADH, along with 340 nm absorbance, is proportional to lactate levels. Thus 340 nm absorbance is directly proportional to lactic acid concentration.
The protocol procedure includes a single read step at 340 nm. A linear regression curve fit is used to determine unknown concentrations.
The experiment file contains the absorbance determination, which is used to plot the standard curve.
The plate layout includes 12 standards in duplicate, in well positions A1 through B12. The standard concentrations range from 0 to 40 μg/ml. 36 unknown samples are defined in on the plate.
The standard curve is reported along with equation parameters and correlation coefficients. A matrix report is configured to provide the (1) 340 nm absorbance and (2) calculated lactate concentrations. A column type report containing the statistics for standards and samples is also provided.