Dear All,
I am testing out an europium labelled peptide for the ligan binding assay and was hoping to find the Kd through the saturation curve.
Can people explain to me why my saturation curve is a straight line rather than what it should look like.
Thank you
It looks like you haven't used enough ligand to saturate the binding system. If it's not too expensive, try using 10x, 100x ligand. Also you probably expect a sigmoidal curve, and that requires on a log x axis.
its difficult to answer without axis molarity definition. It seems to me that the second it is not a logarithmic plot. Transforming in a real Log scale the vertical axis your curve changes a lot.
The classical approach is to transform the values found in the first plot in the fractional alfa values ( I-I final/ Ifinal-I init) for every value of Ligand (L) where I is the values of the experimental point.
Assuming that the bound ligand concentration is negligible with respect to L the equilibrium of dissociation is boundP = (P°-L)/ (Kd+L).
if Alpha = boundP/ P° the equation becomes alpha = L/( Kd+L)
now with some simple passages
(alpha-1) alpha = KdL
This plot in function of L gives the Kd as slope of the curve.
best regards
Sorry there was small mistake ,
please correct
as follows
"now with some simple passages
(alpha-1) / alpha = KdL
This plot in function of L gives the Kd as slope of the curve.
best regards"
Hello! I think there is a biacore T-100 or (3000) at your university. This is good technology and if what are binding is large then it should work quite well. If both of the species are small then maybe not so much. Good luck. Here is the potential lab that has the instrument. https://espace.library.uq.edu.au/view/UQ:722458/UQ722458_OA.pdf
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