I've plotted my data and it looks to be good for a random sequential mechanism. When plotted in excel it fits this trend nicely. However, when I plot these in SigmaPlot, it does not want to put the lines in the right place! I don't know how to manipulate the software so that the graph is at least near to the data! It says that it is a good fit, but visually it is way off. Please see the attached image.
Thanks!
I am not sure why the lines are not closer to the data points, I'd try to plot a single experiment (data points only) and then go Graph -> Linear regression, select both "Each curve" and "Extend to axes", leave "Order" at 1. Then see what happens. You can then add the experiments one by one, using Graph -> Add plot. If that doesn't help, I'd need the original data for further diagnosis.
However, there are several problems here that I can identify:
First of all, although Lineweaver-Burk-plots are nice for showing the result of an experiment, they should never be used for estimating the parameters, as the transformation also affects the errors of the data points and hence affects one of the conditions of line fitting, homoskedasticity (more simply put, Delta y becomes dependent on x).
Sigma plot offers the option of Marquard-Levenberg fitting the curves directly (Statistics -> Regression wizzard, select equation category "Hyperbola" and Equation Name "Single Rectangular". Then follow instructions of the wizzard.
The second problem is that you have too few data points, for each curve you need at least 12 substrate concentrations, evenly spaced from 0.1 to 5 Km. This rule applies to straight LB-plots, for curved plots you need to increase data range and number of points. Your experiment, however, does give you a nice initial estimate of Km for planning the "real" experiment.
The third problem is that your data apparently do not follow the Henry-Michaelis-Menten formula, they appear curved in the LB-plot. You may be looking at a case of substrate inhibition, but to sure requires data at higher concentration.
You can adjust regression line by Statistics - Regression Wizard for each plot. But, if you used the option Graph - Linear Regression you must uncheck the box "Pass through origin".
I agree with Marcia answer ..you can use Statistics - Regression Wizard for each plot.
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