I want to use it as a co-factor of an enzymatic reaction, to evaluate the enzymatic performance of the enzyme and the kinetics of the reaction. I have read that it can be very tricky to work with NADH as it is hygroscopic, can oxidize and can form inhibiting compounds when dissolved and stored. Is there perhaps any NADH that is sold in solution?.
I have used the powder NADH sold by Oriental yeast Co. ltd. without any trouble. To my understanding NADh can be unstable on solution, so I try to make it fresh every time I do an assay.
However, if you are having troubles an alternative assay could be using NAD+ with glucose and glucose dehydrogenase to make NADH and glucoronic acid. Yor can have a measure glucoronic acid formation.
Sigma offers pre-weighed vials of NADH. They can be stored at room temperature and reconstituted in assay buffer just before use. Very handy if you can afford it. Otherwise, make a fresh solution just before use each day and keep it on ice.
Hi!
Its not so difficult to work with NADH. It is hygroscopic. But its always better to prepare fresh everytime you need. Or else, when you prepare your NADH solution at that time only you divide it into different aliquots and store them in freeze. When you want to use it, take it out some time before you are preparing your assay mixtures to get it normal and use it. But you should not store it long in solution, may be for a month or two. Please check its stabilityin solutions/ buffer, you are usin at web.
Thanks.
Puja
Hi Kelly. I use NADH disodium salt from Roche (catalog number 10 128 023 001) for several enzymatic assays. I prepare the concentration I need, store in small aliquots at -80oC and can keep it working very well for some months.
Depending on your protocol, the liver NADH works fine as well and can extract and use it fresh and be very effective but need to to make sure that the temperatures are kept low at all times to avoid denaturing.
freshly prepare ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form....
Thanks! I have used the vials Adam suggested. It is indeed very practical. I am using it fresh every time. So far it works very well.
I also use it fresh every time, but i need another alternative as i need to weigh about 0.8mg every time, for making a stock of 5mM of 250ul, out of which i only need 6ul for each reaction of 200ul. So i was planning if i could make a stock and use the aliquots. Otherwise, it leads to wastage of NADH, as out of those 250ul i need only 120-150 at a time. The rest i discard. I do not possess that kind of balance that can accurately measure 0.8mg and it can lead to error in calculations and amount that i can vary. So i was planning to make 1 ml and keep aliquots of 150ul in 7 different vials, store them at -80 or -20, then thaw it in ice and use one aliquot only once..is it fine that way? Please comment
You can use NADH from Sigma-Aldrich, but prepare fresh every time.
Purchase NADH from Sigma and prepare its solution fresh every time you use in Enzyme assay.
Verify the concentration of unoxidised cofactor, NADH, by measuring the Abs at 450 nm. Then you can adjust the volume of the stock solution you add to the reaction. Works on Fresh solution and aliquotes stored froozen. Fresh Solutions are better but often you need only small amounts and then you have to make aliquotes. Avoid to repetedly freeze/thaw the aliquotes.
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