Lactate Dehydrogenase Assay
Method: The reaction velocity is determined by a decrease in absorbance at 340 nm resulting from the oxidation of NADH. One unit causes the oxidation of one micromole of NADH per minute at 25°C and pH 7.3, under the specified conditions.
Dissolve at 1 mg/ml in 0.2 M Tris⋅HCl buffer. Dilute enzyme prior to use to obtain a rate of 0.02-0.04 ΔA/min. in Tris buffer and keep cold.
Determination of Protein Concentration:
Set spectrophotometer at 340 nm and 25°C.
Pipette into cuvette as follows:
Incubate in the spectrophotometer 4-5 minutes to achieve temperature equilibration and establish a blank rate, if any.
Add 0.1 ml of appropriately diluted enzyme and record ΔA340/min from initial linear portion.