Anti His methodology reference flow cell prep

Hi arnoud/Fellow mates

I am using Anti-His AB to capture his tagged receptors. I have the following question:
Do i immobilize the reference flow cell with Anti-HIs as well and not flow the capture in it but only the analyte? or should the ref. flow cell still be just activate deactivate with EDC NHS and blocked! and active flow cell can be Anti-his immobilized?

Thanks
Best regards

Hi biaenthu,

I would immobilize the anti His on reference (ch1) and active surface (ch2). Capture the His-tagged protein on ch2 only and use ch 1 as reference during analyte injections. In this way the surface differences are minimilized and if there is non-specific interaction with the antibody the reference will compensate better.
Do not forget to add buffer only injections for double referencing.

Regards

Arnoud

Thanks arnoud! i did that today and it worked well
So a new challenge that i am facing is working on receptors which have fast dissociation kd 1.6s-1.. it is outside instrument detection limits... but i have to design in such a way that kinetics come out reliable... any input!? on these high kd issues?

Hi, I am glad it worked!

On the following page are two things you can combine.
www.sprpages.nl/sensorgram-tutorial/a-curve

At the end there is some text about fast on and off interactions. If you can combine this with equilibrium analysis you can at least determine the KD.

In a T200 you can also set the sampling rate to 10 HZ increasing measuring points and use them to try a normal fit.

Regards

Arnoud

well, the sampling rate is already at 10Hz, and as for the equilibrium analysis is concerned I have tried that... That seems to be fine... but the Rmax is way higher in the equilibrium analysis as well as in th kinetic mode... I guess i need to work on saturating the surface.. so that atleast once curve reaches Rmax! and then take the kinetic parameters into consideration!