Analyte binding to chip surface

Hi. Firstly, I must thank you for making such a helpful and informative site. I have learnt almost all my basics about SPR after reading this website. Infact till now, I have missed this forum!!
I have 2 questions-I am using a SA chip to which I bind my ligand that has a SBP tag.
The analyte that I am trying to bind with the ligand
binds to the matrix and therefore the RU's in both the reference flow cell and the test cell (to which the ligand has been bound) for the analyte are the same. The conclusion is that the analyte is
not binding specifically to the ligand. I have tried adding the NSB reducer from BIAcore,
increasing the salt concentration in the buffer used to dilute the analyte,
checked the pH of the running buffer (i.e greater than the pI of the analyte). I
>know these proteins interact because I have done pull-down assays, co-IP, etc
>to confirm the interaction before I went on doing the SPR. Do you have any
>suggestions on how to solve this problem?
The second question is that when we use SPR to calculate stoichiometry using Rmax values, can we use the Rmax values of the analyte calculted in a kinetic experiment?
Thanks.

Hi,

I'am not familiar with SBP. You do not tell if you can capture/immobilize your SBP-protein to the SA chip. Is it?

this ref may be of help:
Anal Bioanal Chem (2006) 386:13211326

If it is the dextran matrix which is the problem you can make your own SA chip with the C1 (no dextran) sensor chip.


The stoichiometry depends on your ligand immobilization (RU) Rmax (RU) of the analyte injections and the Molecular weight of both.

s = (RU-analyte x Mw-ligand )/(RU-ligand x MW-analyte)

The Rmax should be calculated from a analyte concentration series with concentration up to 10-50x KD. You should check that the actual Req is reaching at least 90% of Rmax.

Arnoud

SBP is Streptavidin Binding peptide and this tag can be used to capture the SBP-protein on the SA chip. I did go thru the reference you suggested, but it did not solve my problem.

1. I tried capturing the ligand on the CM4 chip, but once that binds to the chip surface, it does not bind to analyte (which has been shown to bind this ligand using different techniques)!! It is hard to imagine that all the ligand molecules bound to the chip surface are in the wrong orientation. Can you suggest a reason for non-binding of the analyte and a possible solution?

2. Also, in another experiment with a different ligand and analyte, I see interaction between them, although they do not show interaction when immunoprecipitation experiments are done with mammalian cells. The ka values are in the range of e-3. Can the ka, kd, KA or KD values obtained in an SPR experiment be correlated to in vivo observations or to determine the authenticity of interaction?
Thanks.