I have encountered this article:
http://www.jbc.org/content/276/38/36000.full
which is detailed but to me incoherent - I do not understand which material comes when and the order of the procedure is confusing as well.
I have tried a commercial kit but the results were very poor, I think that the instructions for the preparation of the samples were poorly written. I would rather try doing things again, so if anyone can help I would very much appreciate it.
Hey
Try this article that is based on plant samples : http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/17288982
or mine: http://www.ncbi.nlm.nih.gov.gate1.inist.fr/pubmed/22268572
good luck
Hope this will help you. Know that pyridine nucleotide or other redox compounds are extremely labile and you have to work at 4°C, especially if you want to determine the redox balance.
If you have a good protocol for a NAD cycling assay (we use ADH linked to MTT) then it is just the extraction that changes - acid for NAD+ and alkaline for NADH. That takes out lability issues.
Rich
Just to let know that I used the following method:
Kato, T., Berger, S.J., Carter, J.A., and Lowry, O.H. (1973). An enzymatic cycling method for nicotinamide-adenine dinucleotide with malic and alcohol dehydrogenases. Anal Biochem 53, 86-97.
http://www.ncbi.nlm.nih.gov/pubmed/4351123
I have w written protocol based on this method if anybody needs...
I used a calibration curve with known concentrations of NAD/H and normalized to protein concentrations in the sample.
Does anyone has experience with the measurement of NAD and NADH in the mitochondria?
Hi Maayan,
Were you able to make the protocol work ? What was the % recovery you got after spiking the samples with known amounts of NAD+ or NADH?
I have tried the NAD+/NADH kits from Amplite and Biovision, but I am not able to recover the spiked NAD+. The maximum recovery I had was 46% (though for NADH I got a recovery of 96 - 100%). Would you mind sharing your protocol with me, so I can compare it to mine?
I will be working with primary microglia, primary astrocytes and whole brain tissue from mice. I have not been able to detect any NAD+ in the primary microglia and astrocytes so far.
Many thanks,
Gagan
I see that this still gains interest so I uploaded my protocol to the methods section of the site:
Method NAD + /NADH quantification protocol
and I attach it here as well
Happy holidays everyone :-)
Simple method to sense NAD+/NADH in plasma:
Protocol: https://static-content.springer.com/esm/art%3A10.1038%2Fs41598-018-34350-6/MediaObjects/41598_2018_34350_MOESM1_ESM.pdf
Paper: Article The nanomolar sensing of nicotinamide adenine dinucleotide i...
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