I have been studing affinity kinetics of enzyme substrate on BiacoreX100. Everything was perfect when all of a sudden it BiacoreX100 started giving these weird dipping curves. Please see the attached file.
I really appreciate any help or suggestions.
Thank you and Regards
The phenomenon could be due to impurities in the analyte. When smaller compounds bind stronger and replace weaker larger molecules the signal will get lower during the association phase.
Maybe your analyte sample is degraded.
An other explanation could be that the sample plug is mixing with the the flow buffer and therefore lowering the analyte concentration during the injection. You can check this by injection of a solution with 0.2 M NaCl. The curve should be block shaped if the sample is properly separated from the flow buffer.
Yes, just 0.2 M NaCl to check if the injection system works properly.
I did some simulations with BiaEvaluation. I will give the parameters so you can try it your self. Model: Heterogeneous - competing
Both analyte species with 10, 20, 40, 80, 160 nM
Analyte 1 50000 Da, Analyte 2 10000 Da, Rmax 100
ka1 1e5; kd1 0.01; ka2 1e5; kd2 0.001.