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.


  • 0.2 M Tris⋅HCl, pH 7.3
  • 6.6 mM NADH in above 0.2 M Tris⋅HCl buffer, pH 7.3
  • 30 mM Sodium pyruvate in above 0.2 Tris⋅HCl buffer, pH 7.3


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:

Tris⋅HCl, 0.2 M pH 7.3 2.8 ml
6.6 mM NADH 0.1 ml
30 mM Sodium pyruvate 0.1 ml

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.



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