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repeatability issue in data
- shubhamG
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3 years 4 months ago #1
by shubhamG
repeatability issue in data was created by shubhamG
hello, i am working on small molecule and Protein interaction along with protein specific substrate. As we have calculated the KD value of small molecule which is approx 1-10 uM. and we are getting around 64 uM which is way more higher than calculated one.
the concentration range i used is 50uM with 2 fold dilution till 10 points (Triplicate). But now i am not able to generate same data again. I have checked stocks twice, i prepare buffer fresh everyday. The only change is chip with different immobilization level. Can it be possible because of the change in immobilization level (old chip ~4000 RU and new chip ~9000 RU) ???
I also want to know that is it possible that along with the concentration range our KD may also move???
the concentration range i used is 50uM with 2 fold dilution till 10 points (Triplicate). But now i am not able to generate same data again. I have checked stocks twice, i prepare buffer fresh everyday. The only change is chip with different immobilization level. Can it be possible because of the change in immobilization level (old chip ~4000 RU and new chip ~9000 RU) ???
I also want to know that is it possible that along with the concentration range our KD may also move???
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- Arnoud
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3 years 4 months ago #2
by Arnoud
Replied by Arnoud on topic repeatability issue in data
How do you calculate the KD of an interaction? Is it from a previous experiment?
If the binding is a nice 1:1 interaction, the immobilization level should not change the outcome of the kinetics. But when mass transport and rebinding occur, ka and kd change and so the calculated KD. The kinetic constants ka and kd can be calculated differently when the concentration range is not wide enough (0.1 x < KD < 10) but in general when you have one or two points above 1x KD and curvature in the lines this should not pose a problem.
Please show us some sensorgrams.
If the binding is a nice 1:1 interaction, the immobilization level should not change the outcome of the kinetics. But when mass transport and rebinding occur, ka and kd change and so the calculated KD. The kinetic constants ka and kd can be calculated differently when the concentration range is not wide enough (0.1 x < KD < 10) but in general when you have one or two points above 1x KD and curvature in the lines this should not pose a problem.
Please show us some sensorgrams.
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