Actually I want to check the level of phospho AKT in wt and resistant PC9 following treatment with gefitinib. I tried with 24 and 48 hours gefitinib treatment and the result was, phospho AKT could not be maintained even in the gefitinib resistant cells with this time period.
So what can I do to improve my result..??
6-12h, because too long time the cell will become very weak and total proteins are commonly dicreased
Since the activation of kinase by phosphorylation is transient, you can do time points starts earlier after minutes and lasts for few hours.
I would try a time-course of 10, 30, 60 and 120 min and check the activity of pAKT and AKT.
Best,
Zack
Signal transduction is a rapid event with kinases being activated very early following cell stimuli. I would conduct a time course starting at 5 min and move up incrementally to 24 hours. Make sure you detect phospho versus total AKT, to ensure that your protein isn't being downregulated or degraded.
Hello Subodh,
gefitinib, by inhibiting EGFR, should lead to de-phosphryloation of AKT in EGFR-mtated cells. So in your untreated cells you should see pAKT and in the treated it should go down or vanish depending on the gef. concentration. In the resistant cells (if the mechanism of resistance is a sec. mutation in EGFR) pAKT should be detectable with or without drug (of course very high concentrations of gef. may still inhibit EGFR/AKT).
So, as I see it, the effect your describing is exactly what is supposed to happen, at least in the wt, after 5min up to 48hrs. In the resistant, check your EGFR secondary mutation and the dose of gef. you're using.
I.e. if by wt you mean "normal" PC9 which have an activating EGFR mutation.
I hope I got your question right and this all wasn't too confusing.
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