I am working on some new anti-cancer drug. In my in vitro experiment, After certain time point treatment of my drug I see obvious decrease in the level of protein AKT,pAKT and p-c-raf by western blot in a dose and time dependent way.Of course the beta-Actin control is equal. I want to ask is anybody have any ideas about what is the most probably reason to explain their decrease. And what's the most probable target my drug functions on. Or maybe give me a little bit hint what should I explore the next step.
Thank you very much for every of your answers in advance!
interesting, I would explored the transcription level of AKT and downstream target of the AKT to see the effect of your drug on the downstream genes and or proteins.
good luck
Hi Jun Xing,
Generally pAkt levels were normalized with Akt. But in some circumstances like in your case were basal levels also changes(which is quite surprising), we check for actin levels also to confirm changes. If you inhibit protein translation mechanism(may be your drug targeting one of the machinery of it) like AMPK targets can inhibit both basal and phospho forms.
Since you are using anti-cancer drug, leads to ROS generation by which many pathways can be explored. All the very best.
It would be good to ponceau stain the membrane to see if total protein levels are reasonably unaffected. Actin alone is generally a bad proof of equal protein loading, especially if bands are saturated/outside of the linear range.
Try to co-treat with proteosome inhibitor. if you still see the decrease - your drug affects the synthesis rate (at any point: transcription or translation). if you manage to stabilize your targets by this treatment -- the drug is accelerating degradation.
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