SPR on Kinases

I have following problem at hand. I would appreciate if other more experienced users could help.

The instrument in use is SensiQ ( & not Biacore) which has very similar platform and running operations.

The ligand is a Kinase which is a homodimer (40 kDa). The binding site is at interface of two monomers and require ATP/Mg ( as per crystal structure) pre-bound to be able to bind to inhibitor compound. The protein is His tagged and has little drift once immobilized on Ni-NTA chip. The running buffer and sample buffer is same ( 100 mM Hepes pH 8.0 , 150 mM NaCl, 0.01 % Triton X-100,2 mM ATP, 10 mM Mg ,DMSO 2% (V/V)). The analyte MW is 300 and it has ~3 nM IC50 based on enzymatic assay. Protein immobilized at 1100 RU on channel 1 and about 2500 on CH 3. Instrument was cleaned thoroughly before run and all buffers made fresh and degassed.

Problem 1 : Inverted curves : Could these be due to compound or ATP/mg interaction with reference channel ?

Problem 2: very low signal ( Rmax at 10 RU) from compound interacting with protein compared to buffer injections.

Problem 3: Channel 1 where protein is at lower level (1100 RU) has positive response curve for blank injections and CH 3 (2500 RU) has negative response curve.

Question : for such kinases is it important to have immobilization performed using buffer containing ATP/Mg ?

Any comments ??

Thanks.
Aman

Problem 2: very low signal ( Rmax at 10 RU) from compound interacting with protein compared to buffer injections.

Answer: Calculation of the Rmax (Ligand 40.000 Da, Analyte 300 Da, 1 binding site) gives 10 or 19 RU for an immobilization of 1100 or 2500 RU. So I think that your Rmax measurement is ok. You can try to capture more but the stability of the surface will probably drop because there are less free NTA sites left.

Problem 1 : Inverted curves : Could these be due to compound or ATP/mg interaction with reference channel ?
Problem 3: Channel 1 where protein is at lower level (1100 RU) has positive response curve for blank injections and CH 3 (2500 RU) has negative response curve.

These negative signals are always puzzling.
-Can you tell what you inject during a blank injection?
-Is the raw sensorgram already a negative curve or only after subtraction?
-Maybe you can share some figures to show what is going on?

What can be a problem is the difference between reference and active surfaces because the protein density is different. Especially with DMSO in the buffer which tent to give response differences on protein density.

This article may be of help :
www.sciencedirect.com/science/article/pii/S0079610714001102

Question : for such kinases is it important to have immobilization performed using buffer containing ATP/Mg ?

Answer: The Mg could compete with the Ni but since you have decent capture I don’t think there is a problem with having ATP/Mg in the buffer. Unless you want to investigate I would leave it in the buffer.

Kind regards
Arnoud