Which kinetic data between 37 degree and 25 degree to trust - Forum


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Which kinetic data between 37 degree and 25 degree to trust 16 Nov 2021 23:12 #1

Hi Folks,

Situation: Very slow off rate binder with koff <1e-4 or -5. KD in low nM to fM range around LOD of S200. Testing using single cycle kinetics, low target density, Rmax about 10-15 RU using reversible biotin capture CAPture kit, at 25 degree (with top Analyte conc ~ 1uM) and then at 37 degree ( with top analyte conc ~5 nM, longer contact time and dissociation time). I got two sets of kinetic data with clear dissociation at 37 degree vs almost no dissociation at 25 degree. Should the data at 37 degree be only used as validation for data at 25 degree or should 37 degree data be the primary data for future SAR ?

37 degree: ka 4 X e6, kd 3 X e-3, KD 0.8 nM, Rmax 16 RU, Chi2 0.06
25 degree: ka 9 X e5, kd 3 X e-4, KD 0.4 nM, Rmax 10 RU, Chi2 0.07

Feedback highly appreciated
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Which kinetic data between 37 degree and 25 degree to trust 17 Nov 2021 19:36 #2

Hi,
One of the parameters kinetics depends on is temperature. As you can see in your data a higher temperature gives a faster association rate ánd a faster dissociation rate. So there is no right or wrong answer here. Kinetics should always be reported with the used temperature and buffer conditions.
In that respect please read the article about the KD (www.sprpages.nl/equilibrium-dissociation-constant).
The temperature dependency can be exploited: see Navratilova (www.sciencedirect.com/science/article/abs/pii/S0003269707000486)

Kind regards
Arnoud

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