Doubt for Immobilization

Dear team,
Please help me increase my knowledge and troubleshoot the following issues:

1. During amine coupling immobilization of Thrombopoietin receptor:
Under these conditions, the immobilization injection reaches approximately 190 RU, but after the post-ethanolamine injection, the final response exceeds the target by up to 100 RU, showing 300 RU of immobilization.
Method- AMINE COUPLING
Target: 200 RU
Concentration used: 2.0 µg/ml
Immobilization buffer: 10 mM Acetate, pH 5.0
SPR Model: Biacore 1K
Why does this happen?
What can be done to reach the target?
Should I increase the target value?
2. In one of the performed experiments, why does the immobilization show "out of ligand," while the final response shows 240 RU, which is above the target?
3. In multiple immobilizations, the bound response is near the target level (i.e., 200 RU), but the final response exceeds the target by 90-100 RU, reaching approximately 300 RU. Why is this happening?
4. I have tried acetate pH 4.5 and 5.5 as well to know the electrostatic interaction between ligand and surface. But it results are random and it does not give a decent response.
5. I have also performed immobilization by specific contact time of 420 sec using 1ug/ml of TPOR on CM5 chip. First it reaches approx. 250 RU or 270RU for response bound and then final response bound comes to 210 RU or 199RU.
Please suggest what should be optimized to achieve proper immobilization and how should I proceed with.

Thank you

First: You must know that the immobilization is not that precise procedure. There are many parameters and different chemicals involved. Effects on the matrix and the immobilized ligand can alter the response levels significantly.
Second: the algorithm of the instrument tries to get as close as possible to the target response by adjusting the number and injection times. The injection has a certain amount of volume and if the target is not reached within the volume you get out of ligand. Also, when the algorithm detects that immobilisation is not feasible it halts.
Third: immobilization needs a bit of experience and flexibility from the researcher. That said you can steer a lot by knowing what to do. Start with www.sprpages.nl/immobilization .
So, pre-concentration is important. I recommend having a preconcentration ‘speed’ between 20 – 50 RU/s. This wil give you the best control over the immobilisation level. You can achieve this by adjusting the immobilisation pH and/or ligand concentration. The immobilisation itself is a multi step process. Activation determines the number of possible immobilization places. The actual ligand immobilisation consists of the electrostatic pre-concentration step and covalent binding. These are connected but not at the same speed. When the ligand injection stops, non-covalent ligand will wash off. The same is during the deactivation step lowering the resulting immobilisation level.
      
1.      The 1M Ethanolamine could have an effect on the matrix and ligand.      
2.      Interestingly but without the actual sensorgram hard to explain      
3.      See 1.      
4.      –      
5.      Sounds logic, see above. And a drop from 250 to 210 is not much.

My question: how important is it to have 200 or 300 RU of ligand on the surface. How are the analyte injections. Is the interaction giving a good response level? Please show a sensorgram.
Kind regards
Arnoud