variation in kinetic parameters

Hi arnound,

I get a very high variability on the ka, kd parameters of kinetic evaluation (>30% CV across 6 replicates) in Biacore. i do not get that variability in other interaction that I see. the maximum varaibility that I see is in ka. By looking at the curves I feel they very well overlap on each other and I dont see a discernable differences in the same. But I do get ka of 1 log order difference (+5 vs +6) is there anything that I am missing in my method development? any inputs to tackle this variability and get it more consistent?

Thanks
Best regards
Biaenthu

Hi Biaenthu,

I had to think about this because there are very few comparisons of results.
The problem with the CV is that it is sensitive to the values.

With 6 replicates, do you mean 6 independent experiments or six curves in one sensorgram?
You should analyse all curves from one sensorgram globally.
Main errors in kinetic variability is the analyte concentration. Dilution errors and analyte degradation after thaw, - freezing cycles can have some effect. Liagnd integrity (degeradation) should not have a large effect but check the Rmax between the 6 replicates/experiments.
In addition, make sure the buffer concditions are the same.

I have two publications with a benchmark to compare with your experiments.

Navratilova, I., et al. (2007). "Thermodynamic benchmark study using Biacore technology." Analytical Biochemistry 364(1): 67-77.
Rich, R. L., et al. (2008). "A global benchmark study using affinity-based biosensors." Analytical Biochemistry 386: 194-216.

Kind regards

Arnoud