Flowing analyte simultaneously over different immobilized channels

Hi All,

I was wondering if people have immobilized different ligands on a sensor chip (e.g. L1 on C2, L2 on C3, L3 on C4, and using C1 as the blank channel for reference subtraction) and then flowed analyte over all four channels. Will this give you accurate binding data for all ligand:analyte interactions? Expecting the analyte to bind to all 3 ligands (with varying KD values). My thoughts are that if you inject 10 µM analyte and it binds to L1 on C2, then the effective concentration over channels 3 and 4 will be less than 10 µM and therefore give inaccurate binding data. Was wondering if anyone has experimented with this method design and can share their thoughts.

Hi,
We do this often because it gives you three measurements each analyte injection.
Some things to think about:
-keep the ligand immobilization low and therefore the analyte consumption per channel.
e.g. Rmax per channel <100 RU
-keep flow rate high (>30 µl/min) to avoid analyte depletion
also important to lower rebinding during dissociation
You can test for mass transport / depletion by a series of injections at different flow rates (30, 60, 100 µl/min) and compare the curves.
-one regeneration condition for all three ligands can be challenging

Kind regards
Arnoud

Hi Arnoud,

Thank you for the information, it is very helpful.

So just to clarify, in your experience as long as you keep ligand immobilization low (to limit mas transport and analyte depletion) and keep the flow rate high then the three measurements for the analyte injection should be accurate and reproducible?

Many thanks,
Ben

Hi Ben,

The binding and fitting results should be reproducible even when you have three surfaces in series. The problem is in the word accurate, how do you know it is accurate? And how accurate do you want to be (purpose of experiment?) If you are planning to have absolute numbers for the kinetics than you have to do a lot more work than one experiment. Ideally – to get values for ka and kd with standard deviation – the experiment is repeated with different batches of the same ligand and analyte. This means that you have to make several sensor surfaces which gives you the possibility to randomise the ligand location on the chip.
Also keep in mind that differences in buffer composition and analysis temperature have influence on the kinetic constants in case you want to compare them to known values from other experiments or literature.
Kind regards
Arnoud