I am using a lactate assay kit from Sigma Aldrich to measure lactate production in cell culture. But i am having trouble creating the standard curve firstly. I used 5mM lactate with 100mM NAD+, so there is an excess of NAD+ as well. There is no colour change observed in expected wells and the absorbance values are also not as expected according to the amount of lactate added in. I have tried to use the dye, and cofactor from the kit with NADH instead to see if the problem lies in the dye conversion, but there is appropriate colour change and expected absorbance values. This leads me to believe the problem has to be during the conversion of lactate to pyruvate by LDH. But i am not very familiar with this and not sure what is the reason.
You did not mention adding lactate dehydrogenase, which is necessary for the Sigma-Aldrich lactate assay kit I am familiar with.
Gordon L Warren Thank you for your reply. I added LDH in my wells, but there still wasn't expected colour change.
Did the standard curve assays (those with known amounts of lactate) work?
By the way, 100 milimolar seems a huge concentration of NAD+.
I have figured out the problem. It was an oversight where I did not balance the pH of NAD+ i was using. The pH must have denatured the enzymes in my assay leading to the results not turning out as expected.
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