I would like to know the treatment condition for KT 5720 PKA inhibitor for fibroblast cells. Earlier i have used 5um concentration for 16 hours.
Hi Mansoor, I have tried using KT5720 on acute slice preparation. I have used 1uM concentration and used 30 min pretreatment and 15 min treatment condition and it has worked well in my case.
is that sufficient to see the effect of PKA inhibition on my protein of interest? i doubt that. I have come through papers where people used 16 hours or more. I would like to inhibit the PKA activity completely. But i can try 30 min condition as you said. Thanks kaushik.
Hi Mansoor, see the following links.
http://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2003.02076.x/pdf
http://www.jneurosci.org/content/22/12/5016.full.pdf
They have used 1uM and 200nM concentrations of KT5720 to inhibit PKA activity. There are a number of such papers that have used it in these concentration range, although even 5uM concentrations for 30 min also seem to work well. Many such studies have been done on slices, where there are a number of factors that need to be taken into consideration, due to the thickness of the slice preparations.
May be for your system, you can do a time course study along with using some kind of positive control wherein you can be sure that your inhibitor is working well. If you are still not able to get any changes, try using a fresh aliquot (as I did face a similar problem with old stocks of inhibitor - check expiry. Using more recent stocks and vials of the inhibitor helped me solve my problem).
I hope may be this information may help you sort out the problem. Good luck with your experiments.
Kaushik.
http://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2003.02076.x/pdf
http://www.jneurosci.org/content/22/12/5016.full.pdf
thats informative Mr.Kaushik.. my only concern is the limitation of inhibitor that i have. i've used 5um concentration. i cant do time course expt as i mentioned above. But i think i should do that. And yeah i will also use the positive controls when i do my experiment next time. Thanks for your valuable suggestions!!!
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