Orientation strategy question

Hello,

I have analysed a two protein (arbitrarely I call them as Protein 1 and protein 2) interaction in two ways:

First: Protein 1 is immobilized and protein 2 is the analyte

Second: protein 2 is immobilized and Protein 1 is the analyte

In the fisrt case protein 1 is captured by a MoAb

In the second case, protein 2 (which is a protease) is inhibited and tagged with biotin and then immobilized in a streptavidin surface.

The second way give a KD of 5fold the KD obtained by the first strategy.

Is it correct to think that in the second way the immobilization strategy is limiting the Protein 2 mobility (since it is immobilized) and then the affinity increases.

Thanks for your opinion

When capturing a ligand with an antibody you can have two reactions. Look at

home.hccnet.nl/ja.marquar...caying.htm

for an explanation.

In addition, in your second setup the ligand is first inhibited before immobilization. In the first setup you uses un-inhibited analyte. There is a possibility this will give rise to affinity differences.

A 5-fold difference in KD is something but I wonder if it is significant. How hard is this value?

One remark: When immobilization decreases mobility is will decrease affinity -> there are less binding opportunities so you need more analyte so the KD will be higher.

Arnoud, thank you very much for replying.

I am sorry, I wanted to say that with immobilization strategy 2 the affinity may be affected so the KD will increase.

I agree with you that a 5 fold increase in KD should not be significant, this may be relative. But we are worried and we would like to find an explanation.

You say that when immobilization reduces mobility there are less binding opportunities. Please let me ask to you of you know any paper which discuss this reduction in binding opportunities caused by a decrease in ligand mobility.

Thank you very much in advance, your comments are very helpful

It is hard to find publication on this matter but the following four will give some background.

Edwards, P. R., Gill, A., Pollard, Knight DV, Hoare, M., Buckle, P. E., Lowe, P. A., and Leatherbarrow, R. J. Kinetics of protein-protein interactions at the surface of an optical biosensor Analytical Biochemistry 231: 210-217 (10-10-1995).

Edwards, P. R. and Leatherbarrow, R. J. Determination of association rate constants by an optical biosensor using initial rate analysis Analytical Biochemistry 246: 1-6 (1-3-1997).

Kortt, A. A. et al; Nonspecific amine immobilization of ligand can Be a potential source of error in BIAcore binding experiments and may reduce binding affinities; Anal.Biochem.; (253): 103-111; 1997

Johnsson, B., Lofas, S., and Lindquist, G. Immobilization of proteins to a carboxymethyldextran-modified gold surface for biospecific interaction analysis in surface plasmon resonance sensors Analytical Biochemistry 198: 268-277 (1-11-1991).

Ok, thanks again, I am going to read these papers