Dear All
What is the best way to calculate kd (Dissociation constant) for Ligand - Protein binding interactions on Gel using GraphPad Prism.
I have an RNA binding protein that I test for binding to RNA by EMSA. Then I quantify the bands i.e. free probe and the bound complex. GraphPad provides a set of formulas to choose from, whats is the best way to work with it.
Thank you
Hi Ikram, I'm not familiar with Prism, but usually protein binding is either sigmoidal or hyperbolic.
Choose the function that best fits the data.
first plot the data and see the trend of the data (linear, non linear, sigmoidal, hyperbolic etc) then select the best fit model (equations) in PRISM and fit the data to obtain constant value (kd)
Dear Dr Negi
Thank you for your reply. Here is what I do.
1) I plot my data, Protein concentration on X-axis and Bound Fraction (Protein/ligand complex) on the Y-axis.
2) Then I go to the analysis tab choose the Model that best fits my data points and in most of the cases it is sigmoidal, so I select, 'Sigmoidal, 4PL'. This gives me a sigmoidal curve, connecting all the data points. I also get a data sheet, however, that data sheet does not mention any kd. However, if I just look at the graph, I can tell by eye the approximate kd, and in most cases, it is nearly 0.5uM.
3) Then I select the non-linear regression tab and then I get many options to choose from like 'One site-Specific', 'one Site- Total Binding'. I don't really know which one to choose, but if I select 'One Site- Total Binding', I get a data sheet that also mentions kd, however, the kd values are way too high than what appears by looking at the graph e.g. kd 3.6 when on the graph it is apparent to be only 0.5.
I have attached the screenshot in the same sequence as the steps above, in case you want to take a look.
I would appreciate your help.
Thank you
Ikram
Hi dear friend Prism is the best tool for doing this.
I've brought you the path to doing this analysis in Prism 7.
Analyze / Nonlinear regression (curve fit) / Binding - Saturation
I've done a lot of research in this case and Dose Response.
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