Expected response

Hello. I have 2 compounds (A) and (. (A) has Mw of 6000 Da. ( has Mw of 150k Da. I first immobilized A by itself on a chip, FC2. And I got 300 RU. On a separate bench experiment, I combined A and B to form a complex at a 2:1 molar ratio and then immobilized this complex on a separate flow cell, FC3, Assuming my complex has formed into completion, how much increase in RU response would I expect to see in my sensorgram (contribution of compound . Thanks.

Not sure that can be easily calculated. You say that the complex is formed in to completion. So steady state is reached but that say nothing about the amount of complex formed and free A or B in solution. So when you immobilize this solution you fix A, B separately and AB complex to the surface.
To have only complexes on the surface you should make covalent bonds in the complex en purify it before immobilization.

What is your goal?

Kind regards
Arnoud

HI Arnoud, I got more information from submitter. A is biotin-oligo; B is oligo-mAB. The complex (A- will be combined by the submitter in their lab and will give to me. I just wanted to know how much RU increase would the oligo-mAB contribute to A. So for example, if I immobilize A by itself on a Strepavidin surface, FC2, and I got an RU of 300. And if I immobilize the biotin--olilgo/oligo mAB complex on a diferent flow cell (FC3), What would be a theoretical RU increase would I expect that will be contributed by the oligo-mAB to the level of A? I assuming it will be much higher than 300 RU, but not so sure at what levels. Ofcourse, I am assuming that the combination that they will be doing goes into completion. Thanks.

Hi,
What you need is de molecular weights of the compounds since the amount of immobilized ligand (in RU or pmol/mm2) is directly related to this.

As an example let the biotin-oligo: 3000 Da and the oligo-mAB 150000 Da.
When you immobilize 300 RU biotin-oligo then you have 300/3000 -> 0.1 pmol/mm2 of biotin-oligo on the surface.
So to immobilize the same amount of biotin-oligo/oligo-mAB you have to immobilize 0.1 * 150000 -> ~15000 RU.
(ref: www.sprpages.nl/immobilization/immobilization-strategy )

If you can immobilize that amount of biotin-oligo/oligo-mAB complex depends on the crowding on the surface since the complex is much bigger than the biotin-oligo. Maybe it is saver to immobilize the complex first and then the biotin-oligo…
Let me know how this works out.

Kind regards
Arnoud