I am working on SPR T200, I have started following some issues recently as mentioned below.
1. I was working on enzyme-substrate interaction and i got very good slow kinetics and good KD. but recently i have started re-validating my assay neither i am able to see slow kinetics nor good KD.
2. As i am increasing the concentration of substrate KD is also increasing.
3. I have same protein but 10 His-tag which I have immobilized with amine coupling method which also showing very different results. there is no traction between both old and new protein.
4. As I have immobilized 10 His-tag protein with amine coupling method, will his-tag interfere in immobilization? if it will how?
I am happy to discuss over call and skype or through any other medium. Please provide you feedback.
It is difficult to give an answer to these complex questions with the information you give.
1. Can you validate the enzyme and substrate with a different assay than SPR? From what I know is when you use an active enzyme it will process the substrate which will leave the enzyme. This means for SPR that the measurement must be considered with caution because it is not like Langmuirian kinetics.
3 & 4. The 10 HIS-tag should not alter the kinetics (if it is not part of the binding site) or the way the protein is immobilized (amine coupling with primary amines).