Leucine Dehydrogenase Assay - (Jun/08/2012 )

Hi,
Im attempting to generate a leucine standard curve in order to determine unknown sample concentrations. To do this Im doing a leucine dehydrogenase assay where Im using set concentrations of enzyme, NAD and buffer but increasing my concentrations of leucine (conc range: 0-1 uM leucine). Im then measuring the reaction kinetically using a fluorimeter at Abs 340, Ex 460 to detect formation of NADH. The problem is that Ive been so far unable to generate a decent standard curve. The fluorescence fluctuates between each concentration and does not increase proportionally. It should be a fairly straightforward reaction as the substrate:product ratio is 1:1. Im really under pressure to get this to work and I cant figure out why its proving so difficult. Ive done several controls which show that each component is doing what its supposed to be doing seperately but together it does not work. Please hepl!
The assay is a slight variation on the one attached below

3 things:
1) check the sensitivity of the assay, it may well be that you are trying to measure too small an amount of leucine for the reaction to be linear.
2) check the pH of the buffers
3) make sure your NAD is fresh!

Why are you using an excitation at 460 nm? You should be able to just measure the absorbence directly at 340 nm.

you may also be seeing feedback (product) inhibition