Dose response curves

[nico68]

Hello,

I would like to plot dose-response curve but i have a question about it: each point of this curve = one experiment but for each experiment i need to have a stage (saturation) at the end of this experiment or not ? or i take the final concentration of my product at 1 min ?
Thanks a lot in advance.

Nicolas.

[blcr11]

I'm tempted to just say “No” and let it go at that. I’m not sure what kind of experiment you’re doing, exactly. This sounds like enzyme kinetics where you’re measuring the initial velocities as a function of either substrate or enzyme conencetration. I’m not absolutely certain, though. If this is enzyme kinetics, the individual experiments shouldn’t all show saturation, especially at the lower substrate concentrations.

You can do a set of experiments at either fixed enzyme or fixed substate concentrations. At fixed enzyme levels, you would mix different concentrations of substrate with the same, fixed level of enzyme, wait 1 min, and measure the amount of substrate consumed or product formed, whichever is easier. This will be the initial velocity of the reaction in micrmole/min or some such unit. I’m assuming you have a way to either quench the reaction after 1 min, or you’re continuously monitoring the absorbance or fluorescence over time, and you can just “take” the value at 1 min. Adding more substrate should increase the initial velocity up to the point when there are enough substrate molecules around to completey fill all the active sites. Beyond that point, adding more substrate will not change the initial velocity of the reaction. That is the saturation point: the enzyme active sites are fully occupied and you can’t get the enzyme to go any faster.