I am doing aggregation prevention assay of one substrate and a chaperone. the chaperone prevents aggregation upto 90% at 1:10 molar ratio. Now I add a certain inhibitor of the chaperone to see how much aggregation is occuring of the substrate. the negative control is the chaperone-substrate prevention curve and the positive control is only substrate aggregation. for certain compounds I have seen that at various molar ratios like 1:10, 1:5,1:1 the curves are the same, like concentration independent behavior. on going down to 1:0.1( chap-inhibitor), I got the same as the negative control, which means that the compound has no effect. Has someone experienced this before? is it possible theoretically ...I have repeated them and they are long assays as well.
Dear Bhaskar,
When you see concentration independence of inhibitor on chaperone activity, how does the aggregation curve look?
Does the curve look like positive control in the molar ratio range 1:1 to 1:10 (chaperone-inhibitor)? In that case, it would mean that inhibitor is completing inhibiting chaperone activity in that range. You might then have to try in molar ratios between 1:1 to 1:0.1 (chaperone-inhibitor).
Does the curve look like negative control in the molar ratio range 1:1 to 1:10 (chaperone-inhibitor)? In that case your inhibitor might not be inhibiting the chaperone in that range.
If the curves are somewhere in between positive and negative control, then it might be possible that the inhibitor is not able to inhibit the activity of the chaperone completely and is in saturated phase. In that case you should have titration of inhibitor in the molar ratios between 1:1 to 1:0.1 (chaperone-inhibitor).
Best,
Jogender
Dear Bhaskar,
Really interesting!
I would like to know whether you keeping a control of aggregating peptide along with chaperon inhibitor (but no chaperon). Please write me back so that I can provide some more ideas.
Best regards,
Jay
I assume you mean molar ratio of inhibitor to chaperone. If I understand this correctly then below 1:1 you don't get inhibition and above 1:1 you get complete inhibition. This seems like it might be an incredibly strong inhibitor, possibly covalent. In this case then each molecule of inhibitor can completely inhibit one molecule of chaperone.
If it is covalent then you can measure this by mixing your inhibitor and chaperone, then removing the inhibitor (by desalting, dialysis, etc) and then performing the assay - high inhibition indicates a covalent inhibitor. In order to obtain IC50 type values you will need to reduce the concentration of chaperone and thus reduce the concentration of inhibitor giving a 1:1 ratio.
Hope that helps,
Ellis
I dont get complete inhibition. all i am saying is that whatever percent of inhibition i am getting is, above one molar ratio, invariant. So, say, 1:5 and 1:10 the graph is the same. Aneways, thank you all. I got my answer from Mr. Jogender Singh. Thank you again.
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