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• #16988
dalin
Participant

With the fluorometric enzyme Assay how to convert the FLU/min (obtained as FlU/min slope for every concentration of substrate; serial dilutions of substrate at concentrations 200 uM to 50 uM), into to RFU/mg/min of enzyme, that is nessesary to obtain the rate of reaction for the enzyme.
Enz. Eny used is 20 ng
How to obtain the final fluorescent product, by comparing the date with a standard curve of the free cleaved fluorescent in such a way that give RFU/mmole  of product (or any comparable unit).
FLU = relative fluorescent unit.
The substrate is labeled (non-radical) With a fluorescent molecule which once is cleaved the fluorescent intensity can by measured.
Cheers

• #112847
JackBean
Participant

just divide it by amount of enzyme (20 ng)

• #112855
dalin
Participant

Thanks for your respond. But how I convert RFU that is cleaved from the substrate into uM or mmole using a standard curve with the free fluorescent as a standard reference. I am kind of stuck here how to work it out using excel. Is it right if I plot serial concentrations of my reference fluorescent for instance 2 to 15 uM on the x axis and the RFU on y-axis. Then I would say my my slope = RFU / uM
And then if if I put each RFU values that I want to convert them into Concentration using this equation
uM = RFU / slope
Is it correct.
Also, I am not quite sure how to convert uM values into mmole?

• #112857
JackBean
Participant

yes, that seems pretty much alright.

The concentration is c = n/V

• #112873
dalin
Participant

Dear Jack,
Thanks for your reply. But can you give details how to convert concentration for instance in M, mM, uM into moles, mmoles, nmoles as i am kind of "stupid" sometime with the conversion between concentration and other formula. Please detailed example if you like?
also, the n is mole??
Cheers

• #112874
dalin
Participant

Just to also add to this problem!
The concentration in mM is supposed to be obtained by comparing the enzyme data with the standard curve in such a way that I convert the RFU (for the enzyme data) into mM using the free Fluorescent reference curve ( consist of mM concentrations of the Fluorescent compound against their RFU).
1) My issue here or what make it difficult for me – is to convert the those mM into grams, moles, mmoles etc
– what molecular weight i will be referring to? is it substrate (which i doubt it – wrong), or the molecualr weight of the standard reference fluorescent that is used to plot the standard curve? I think it is the molecualr weight of the fluorescent is I am right?
2) what volume i will be using or referring to is it the total volume of the reaction components in each well.
Lets say for the actual enzyme assay I add all my components of buffer, enzyme, substrate, etc and the vinal volume here for the assay is 50 ul. also, the reference compound was prepared for the standard curve in basically the same volume – that is 50 ml.
So, then I can understand that to the equation i am putting the right things
M = mol/ V (in L).
Grams = moles X MWt

• #112875
JackBean
Participant

Yes, n is amount of substance http://en.wikipedia.org/wiki/Amount_of_substance in moles

if you have 0.5 mol in 0.1 l, than the concentration is 0.5 mol/0.1 l = 0.05 mol/l