The percentage of inhibition not only depends on inhibitor concentration and dissociation constant (or inhibition constant). It also depends on protein (cdk1) concentration. For example, if you have a protein concentration of 100 nM, no matter what Ki your inhibitor exhibits, you must add "at least" 100 nM of inhibitor in order to reach total inhibition (if affinity is extremely high, 100 nM inhibitor might be enough, but if affinity is not high, much more than 100 nM will be required for 100% inhibition).

If Ki is equal to 35 nM, and the inhibitor concentration is 35 nM ([I]=Ki), for a protein concentration of 3.5 nM, 35 nM and 350 nM, the percentage of inhibition is 48%, 38%, and 9%, respectively. If the protein concentration is lower, you will not get more than 50% inhibition; and if the protein concentration is higher you might end with no inhibition at all.

If your protein concentration is 35 nM and Ki=35 nM, adding 40 nM or 50 nM inhibitor will provide you with only 42% and 48% percentage of inhibition. Therefore, adding a little more inhibitor might not provide total inhibition.

If you do not know the protein concentration, you will have to rely on trial/error strategy. If you are able to determine the protein concentration, then, you can estimate how much inhibitor you need for reaching a certain percentage of inhibition.

The percentage of inhibition not only depends on inhibitor concentration and dissociation constant (or inhibition constant). It also depends on protein (cdk1) concentration. For example, if you have a protein concentration of 100 nM, no matter what Ki your inhibitor exhibits, you must add "at least" 100 nM of inhibitor in order to reach total inhibition (if affinity is extremely high, 100 nM inhibitor might be enough, but if affinity is not high, much more than 100 nM will be required for 100% inhibition).

If Ki is equal to 35 nM, and the inhibitor concentration is 35 nM ([I]=Ki), for a protein concentration of 3.5 nM, 35 nM and 350 nM, the percentage of inhibition is 48%, 38%, and 9%, respectively. If the protein concentration is lower, you will not get more than 50% inhibition; and if the protein concentration is higher you might end with no inhibition at all.

If your protein concentration is 35 nM and Ki=35 nM, adding 40 nM or 50 nM inhibitor will provide you with only 42% and 48% percentage of inhibition. Therefore, adding a little more inhibitor might not provide total inhibition.

If you do not know the protein concentration, you will have to rely on trial/error strategy. If you are able to determine the protein concentration, then, you can estimate how much inhibitor you need for reaching a certain percentage of inhibition.

Basically the Ki of an inhibitor is analogous to Km for a substrate and thus defining the concentration at which half of the inhibitor forms I-Enzyme complexes and the other half is unbound - so by definition the Ki is not the concentration of full but only half maximal enzyme inhibition (even that is not exactly correct, since half maximal inhibition is defined by the IC50 value since Ki and IC50 may differ depending on the mechanism of action of the inhibitor). If you know the Ki or IC50 of an inhibitor you should use at least 10x that concentration to achive >90% inhibition. Which you can clearly not do with the values for CycA and CycB - CDK1 complexes so close for theRO-3306 inhibitor.

To my knowledge there is no small molecule inhibitor available which is selective enough to block CDK1-CycB completely while leaving CDK1-CycA untouched.

If you require to get rid of CDK1-CycB activity in your lysates you should consider to immunedeplete the CycB and then proceed with your lysates.

I would do concentration gradient,

Thank you very much for your explanations!

N