I have calculated the kd value for peptide (13 mer peptide) and LPS (Lipopolysaccharide) interaction through SPR and fluorescence displacement assay and got 1000 fold difference. the equilibrium dissociation constant value is 15 micromolar in fluorescence titration experiment and 1.3 millimolar (Affinity constant) in SPR experiment . Can anybody suggest why I am getting this difference?
SPR experimental condition
Immobilization buffer-10 mM PBS buffer (pH-6.0)
Running buffer-10 mM HEPES, 150 mM NaCl, pH 7.4)
250 micromolar peptide was immobilized and 49 µM, 98 µM, 245 µM, 490 µM, 980 µM concentrations of LPS were injected and affinitiy constant was calculated for peptide-LPS interaction.
For Fluorescence assay
Tris-cl (pH -7.4) was used
BODIPY-TR-cadaverine (BC) as a fluorescent probe
Increase in fluorescence intensities (at 680 nm wavelength) with increase in concentration of peptides were plotted and the equilbrium dissociation constant (Kd) was calculated using Igor Pro 6.36 (Y = Bmax × Xˆh/(Kdˆh + Xˆh); where X: concentration of peptide (µM), Y: fluorescence intensity (a.u.), h: Hill slope)
You can loose the affinity after immobilization if that leads to covering of the interaction site. I am not sure also about the efficiency of immobilization of such short peptide.
Hello, I will suggest you to try a capture of a biotin version of the peptide (N term or C term) over a SA sensorchip by SPR. The binding site of your LPS seems to be not accessible with your current immobilization.
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