How To: Use the Enzyme Kinetics wizardDocument ID: DQ252
Relevant to following products: WorkOut 2.0
Document description: How To: Use the Enzyme Kinetics wizard
The Enzyme Kinetics wizard is the most complicated of the WorkOut wizards. It should be noted that this wizard is for this very specific application (plotting slopes from kinetic measurements against concentrations to calculate Vmax) and should not be confused with any other type of kinetics assay (which you would use the Generic Wizard for).
To understand what it does it is useful to look at the examples installed into your Results folder. WorkOut installs “Example Enzyme Kinetics.ars” and with build 295 “Example Enzyme Kinetics (2).ars”:
The first example is very noisy data with lots of replicates (so the report does not look that great) but if you select the Analysis tab and then Group Vmax, then press Overlay you can see the results. (Note, you may get some broken links in the report on first opening this example file – just press Recalculate to create the images on your PC)
The second example file contains data which is a lot cleaner and easier to follow, although there is only one sample group. If you go to the Analysis tab you can see the steps of the analysis: first WorkOut calculates the linear slope of each well of the first 1000 seconds (Kinetic Fit tab) and then fits a Michaelis-Menten of these slopes against the specified concentrations (Group Vmax tab). The Vmax, Km and R2 are calculated and included.
In a WorkOut Enzyme Kinetics assay, the Vmax of each sample is calculated. Each sample group is in serial dilutions (each group is diluted in the same way). Each sample is measured repeatedly (kinetics) and it is the linear or exponential slope of each diluant that is plotted against its concentration.
The trickiest part is setting up the microplate layout; the information displayed when the Enzyme Wizard is selected from Create a new protocol in the Organiser describes how to do this. Essentially, you set up each group in the microplate layout editor and define all wells belonging to that group as the same replicate although they will be diluted differently (there must be at least 4 replicates). After setting up the microplate layout editor, the next step in the wizard “Concentrations” allows you to specify the sample concentrations and also the concentration replicates – the microplate thumbnail flashes here to show which concentration you are specifying. As with all the wizards, press the Help button for more information – this describes more about the subscript numbering used in each well.
DQ111: The software shows me the Vmax value but how can I find the Km value?
How To: Find the Km value from an Enzyme Kinetics assay or Group Vmax transform.
DQ125: How To: Find the Rē of the linear fits in Enzyme Kinetics.
How To: Find the Rē of the linear fits in Enzyme Kinetics.
DQ101: How do I find which version of the software I am using?
How To: Find which version of the software is in use.