Just like the ligand, the analyte can give some real artefacts. It is good to be aware of the possible problems you can encounter.
The analyte concentration has a direct influence on the association phase because the equation contains a concentration term. With an actual analyte concentration that is half of the expected value, the ka will be reduced by half and the KD twice as high (as expected). Dilution errors, evaporation of the solution and adsorption of the analyte to the vial wall can cause higher or lower concentrations than expected.
The first runs after cleaning the instrument can suffer from adsorption of the analyte to the tubing and IFC-walls. A pre-run with a high protein solution (for instance BSA) can reduce this effect. When analyte and flow buffers are not matched, drift and bulk effects may cause large residuals (1).
With a real concentration half of the expected the ka will be half lower and the KD two times higher:
|Situation||Conc.||ka (M-1s-1)||KD (M)|
|expected||50 nM||2.75 105||4.42 10-9|
|real injected||25 nM||5.45 105||2.21 10-9|
|No influence on kd, Rmax, Req and Chi2|
And use the correct analyte concentration. Too low concentrations will give low signals but high analyte concentrations (> 100x KD) can give rise to all kinds of non 1:1 interaction effects that cannot be modelled out.
|affinity||0.1 – 10 x KD|
|kinetics||0.1 – 10 x KD||for kd > 10-2 s-1|
|1000 x KD||for kd < 10-4 s-1|
Analyte concentration and injection time should be chosen such that at least some of the curves reach steady state during injection and approach complete dissociation during the dissociation phase. Analysis based on incomplete association and dissociation data run the risk of missing heterogeneous binding behaviour (2).
However, when reaching equilibrium and the signal decreases to the end of the injection, a smaller volume should be used. This is because the dispersion of the sample plug with the running buffer lowering the initial analyte concentration. To check for a uniform sample plug, inject running buffer with 0.25 mM NaCl. A non-uniform sample plug may indicate that the system needs cleaning.
When higher flow rates give higher dissociation rates, this could indicate rebinding effects (3). Rebinding effects may also be suspected when the dissociation does not follow a single exponential curve and baseline levels are not reached.
Low-affinity interactions are characterized by continuous rebinding of the analyte. When only a fraction of the ligand sites are occupied, the binding seems to be more stable than at high concentrations. High concentrations of analyte can give multiphasic binding curves and a drop in binding level during injection phase indicating a less stable binding. In addition, at high analyte concentrations there are fewer free binding sites and thus higher dissociation constants.
To test if rebinding occurs during dissociation, an injection with ligand can be done. When the dissociation rate increases rebinding can be assumed (4).
|(1)||Webster, C. I., M. A. Cooper, L. C. Packman, et al. Kinetic analysis of high-mobility-group proteins HMG-1 and HMG-I/Y binding to cholesterol-tagged DNA on a supported lipid monolayer. Nucleic Acids Res. 28: 1618-1624; (2000).|
|(2)||Roos, H., R. Karlsson and K. Andersson A calibration routine to improve the interpretation of low signal levels and low affinity interactions. (1998).|
|(3)||Nieba, L., A. S. E. Nieba, A. Persson, et al. BIACORE analysis of histidine-tagged proteins using a chelating NTA sensor chip. Analytical Biochemistry 252: 217-228; (1997).|
|(4)||De Crescenzo, G., S. Grothe, R. Lortie, et al. Real-Time Kinetic Studies on the Interaction of Transforming Growth Factor alpha with the Epidermal Growth Factor Receptor Extracellular Domain Reveal a Conformational Change Model. Biochemistry 39: 9466-9476; (2000).|