We have synthesized some inhibitors for PKM2 enzyme. we are planning to perform enzyme kinetic studies. we studied some protocols for the same but conc. of enzymes to be used are missing. some of them suggest varrying enzyme conc while using the same conc. of substrate, while others mention varying substrate conc. Please suggest how to start.
if someone has protocol for same please share.
Since enzyme preparations can vary in actviity, you should figure out how much of your preparation is needed. A good way to get started is to vary the enzyme concentration and measure the product formation as a function of reaction time. The goal is to identify an enzyme concentration that gives a linear increase in product concentration during the amount of time over which you would like to run the reaction (anywhere from 10 to 60 minutes is typical), while making enough product to give a strong signal.
Hi Heena,
Please consider a range of enzyme concentration so you can get a linear curve with good R-value. Then apply the sample readings to the standard curve to obtain the amount of unknown in the sample
Hello, everyone,
how to decide for varying the enzyme con. like which range should be taken. Can we take normal body conc as maximum range and dilute it respectively.
Do you know the normal body concentration of the enzyme?
In any case, I would start by making a set of 2-fold serial dilutions of the enzyme and testing them all to find the right range to work in. The highest concentration might be 1 micromolar.
Use PamGene’s global kinase activity arrays. They are more sensitive (1-5 ug proteins only, 10-50k cells), robust, reproducible (
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