Hi,
I have performed SPR for protein-ligand interaction. I have immobilized the protein on to Chip by amine coupling and used 30uM to 480uM of the ligand for kinetics analysis. However I get all sensogram with same RU. I have also tried lower concentration which does not show binding. Please help me in getting KD value. I have attached the Sensogram.
Thanks in Advance.
Manjula
Hi there,
What is the actual amplitude of the signal? It seems quite low compared to the noise of the baseline.
If you mean 30 to 480 micromolar the ligand cannot be so "bad". You must reduce concentration. If yourbnding is specific it is imposssible that you get no binding at lower concentrations. You must end up with a dose repsonse curve and able to calulate the equilibrium cosntant. On the other hand, what do you use do get dissociation at circa 370 sec? and at what concentration?
Theoretically we assume that all immobilized ligand molecules are fully available and functional for analyte, which is most likely not the case with amine coupling. Binding sites can be affected by the immobilization procedure. Ideally the analyte concentrations for robust kinetic determination should comprise at least five concentrations with at least one of them in duplicate (preferably higher concentration) in addition to a zero concentration sample. At high concentration range the binding curves are clustered together. Make sure immobilization procedure is correct. Purity of the sample is very important (which I am sure it is fine in your case).
Always check buffers-pH, pKa, filterred (according to your ligand and analyte requirement). IF you know there is another better analyte specific to the ligand what you are using here, run 1-2 concentrations of that too. Like specific antibody-antigen.
Regards
Yuvraj
Hi yuvraj,
I have performed kinetics fir other compounds in same chip and I have got the good kinetic constant fir them . Hence I ruled out the problem with biosensor chip. However I have tried the same experiment with two different buffers. But still I got same result.
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