I need help analyzing enzyme kinetic data.
I have data from the Octet K2 system. In my experiment, I load the sensor with our protein of interest (6XHis tag on my recombinant protein to Ni-NTA sensors) and then expose this sensor to increasing concentrations of the candidate binding protein (five concentrations per experiment and each experiment is replicated four times). Each association step is followed by a dissociation step in buffer. A control sensor is used in each experiment where a sensor is loaded with the protein of interest but only exposed to buffer. (See picture, Part 1)
I have separate data where I loaded smaller recombinant domains of the protein of interest to the sensor and exposed it to the candidate binding protein. I would like to combine this data (the binding of the full-length protein and the binding of the domains) on the same graph.
My problem: In trying to analyze the data with the software provided with the Octet system (HT 11.1), the data misaligns. (See picture, Part 2)
My goal is to determine kinetic constants (KD) of the full-length protein and its separate domains to the protein of interest.
Suggestions for correctly aligning the data in the Octet software HT11.1? (I think the misalignment is because the program is trying to align the y axis to baseline 1 instead of baseline 2, which is the baseline right before the association step. If so, can you change this label after the fact?)
If the glitch with the Octet software cannot be fixed, then is there a manual/tutorial for the enzyme kinetic module for Sigma Plot?
I found I can extract the raw data from the Octet system. I can remove the background from the control sensor and manually assign concentrations. I uploaded this into Sigma plot 15, which has an enzyme kinetic module. I found the embedded help guide, but I have specific questions. For example:
*My candidate binding protein does not change, but how do you take into account the change in the kilodaltons of the proteins that are loaded to the sensor, full length vs. the smaller domain proteins? This is automatically taken care of in the Octet software.
*How do I differentiate between the association and dissociation phases?
I am new to Octet biolayer analysis and the Enzyme Kinetic Module analysis in Sigma Plot.
Any help will be greatly appreciated! I am happy to provide any more information.
Hello Jade,
I am not a regular Octet user, so I do not know how to fix the misalignment in Octet software (I'm sure it is possible), but I've done many SPR experiments.
1) You immobilized first the full-length protein, then its domain. In the formulas used for the molecular mass of the immobilized ligand is not used, so it is irrelevant. What counts here is the number of binding sites on the ligand, usually taken as 1 (1:1 binding, which follows a Langmuir isotherm).
2)To manage the required analysis in SigmaPlot or any other general curve-fitting software is possible but can be a headache, not to mention that the fitting needed is nothing to do with enzyme kinetics (maybe there is a binding modul in SigmaPlot, I do not know the current versions). What is more straightforward is to use some curve fitting software for SPR, like Scrubber or TraceDrawer (or the very old Clamp).
Hope this helps. Best: Karoly
Karoly Liliom, thank you for the helpful suggestions and the clarifications!
Having worked with the data before asking this question on Research Gate, I can agree that working in SigmaPlot has been a headache.
I requested a trial of TraceDrawer, and I will let you know how the analysis goes. From what I saw online, this is exactly what I was looking for.
Thanks again!
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