Generating good kinetic data

I am new to SPR and BIACORE and I am learning on the go.

currently using single cycle kinetics:

I have a project that aims to assess the affinity of RGD integrins (and mutants) for their ligand (latent TGF-.

LIGAND: I have taken the approach of immobilizing (via free amines) the integrins and a control at low level (theoretical rmax calc of 100 RUs).


When I pass the analyte over the chip (CM5) I generate response curves and the signal is positive even when I subtract the negative control channel. However, even though we would expect a 1:1 binding model, my data does not fit well at all to the model. in fact the curves do not look great. I feel I need to increase dissociation time and maybe adjust flow rate (currently only 60s dissociation)

I have included an image of my curve shapes, both raw and control subtracted.

How might I best improve upon my kinetic data - assuming sample prep and buffer is not an issue (preps look good on gel and are desalted into running buffer).

Many thanks,

Rob

Hi Rob,

The curves look indeed a little weird. Especially at the start of each analyte injection. The binding is too steep (could be mass transfer but I doubt it). On the positive side the interaction is levelling off (saturating the ligand) and that is ok. Thus a higher flow rate could lower possible mass transfer. A longer dissociation time will not make the interaction more 1:1. Adding a longer dissociation at the end (5 – 10 minutes) may make it possible to determine the dissociation rate constant more precise.

Since the interaction looks strong (low dissociation) you could try to extend the analyte concentration range by making a step 3 or 4 dilution. In this way, the first injections have a low response and you end almost saturating the ligand.

Arnoud

Hi Arnoud,

Thanks for your suggestions! Could it be that I am saturating at the lowest concentration of analyte? I may have made a rookie error in that my lowest concentration of analyte is probably around the KD of the interaction...

Hopefully I will see some improvement by introducing a wider range of analyte concentrations!


Thanks again,

Rob