How to attain a consistant kd values

Dear Sir,

We are facing problem is attaining a consistant kd values. The analyte binding to ligand is too high which is resulting in no dissocuation at all. We tried lowering the pH of the running buffer also but it resulted in proper dissociation but ka values were getting affected from cycle to cycle(Binding values were decreasing). Please help us in working with such method where dissociation is a problem.

Thank you
Best regards

Hi,
A tight binding is always difficult to measure since the incubation times to reach equilibrium or sufficient dissociation can be very long. You can try to measure the dissociation in a well equilibrated system, but when the dissociation rate is below 1.10^-6 s^-1 this will take more than 15 hours to have only 5% dissociation. And you need a very properly matched reference to do the double referencing which means that you also have to run a blank for the same period of time.

I don't think SPR is the best solution when the interaction is very tight (k_d < 1.10^-6 s^-1) and I would suggest to use other means of measuring.

I found the following question on research gate ( www.researchgate.net/post/How_would_you_...igh-affinity_binding ) and the answer of Karl Andersson with the references is a good starting point to solve your interaction.

ITC ( www.biapages.nl/itc-isothermal-calorimetry.html ) is also suggested but this technique uses a lot of sample compared to MST ( www.biapages.nl/mst-micro-scale-thermophoresis.html ) which would my choice.

kind regards
Arnoud