LDH Assay[1]                                20.09.01

Enzyme activity in the sample: 1 mU/ml up to 1 U/ml

 

Buffer

4.92g    Tris Base

5.95g    NaCl

Ad 500 ml A. dest, pH 7.2 with HCl

Reagent solution

297µl    NADH 10mg/ml in Buffer           Roche 128 023

187µl    Pyruvat 10mg/ml in Buffer        Fluka 15990

10.0ml   Buffer

Standards

LDH stock solution                         Roche 107 042

From 1 U/ml always divide by 50% to 2-6U/ml and 0 U/ml

Measurement in Spectralphotometer (λ = 340 nm)

200µl    Reagent solution

14µl     Sample

 

measure kinetic, E340 every 2 minutes for 20 minutes.

Compare with standards (approx 1 mU/ml bis 1 U/ml)

 

Notes

Double concentration of NADH is used compared with the reference

The reaction is started with the enzyme

 

For cell culture medium use a 1:10 and 1:50 dilution.



[1] A. Racher, LDH Assay, in Cell and tissue culture: Laboratory procedures in biotechnology, A. Doyle and J.B. Griffiths, Eds. 1998, John Wiley & Sons: Chichester, New York, Weinheim. p. 71-5.