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Minimum transfection incubation time


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#1 smr86

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Posted 16 September 2009 - 07:36 AM

Hello

I was curious as to the minimum length of time anyone has incubated transfected cells for before analysing them. I will be transfecting SH-SY5Y/Hela cells with an expression vector I created, to check the vector is functioning correctly, but also at the same time treating the cells with IL-1B and essentially I will be doing a western to look at pJNK expression following transfection and treatment. I will be using Lipofectamine for transfections.

Problem is, a postdoc in another lab told me that pJNK will be activated for up to 30min after treatment, and then its expression will just return to basal levels, so if I wait to lyse the cells til 24/48hrs after transfection I will just see pJNK expression but no difference in expression levels as a result of my manipulations.

Has anyone had this scenario before? This is my first time doing these transfections and also the first time our lab has done these experiments so we're kind of new to it

Thanks

smr86

#2 Dr Teeth

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Posted 16 September 2009 - 10:53 AM

Hello

I was curious as to the minimum length of time anyone has incubated transfected cells for before analysing them. I will be transfecting SH-SY5Y/Hela cells with an expression vector I created, to check the vector is functioning correctly, but also at the same time treating the cells with IL-1B and essentially I will be doing a western to look at pJNK expression following transfection and treatment. I will be using Lipofectamine for transfections.

Problem is, a postdoc in another lab told me that pJNK will be activated for up to 30min after treatment, and then its expression will just return to basal levels, so if I wait to lyse the cells til 24/48hrs after transfection I will just see pJNK expression but no difference in expression levels as a result of my manipulations.

Has anyone had this scenario before? This is my first time doing these transfections and also the first time our lab has done these experiments so we're kind of new to it

Thanks

smr86



Why don't you transfect your cells and then, after 24 h, treat with IL-1B for 30 min and then harvest the cells?

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 smr86

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Posted 17 September 2009 - 01:22 AM

Hello

I was curious as to the minimum length of time anyone has incubated transfected cells for before analysing them. I will be transfecting SH-SY5Y/Hela cells with an expression vector I created, to check the vector is functioning correctly, but also at the same time treating the cells with IL-1B and essentially I will be doing a western to look at pJNK expression following transfection and treatment. I will be using Lipofectamine for transfections.

Problem is, a postdoc in another lab told me that pJNK will be activated for up to 30min after treatment, and then its expression will just return to basal levels, so if I wait to lyse the cells til 24/48hrs after transfection I will just see pJNK expression but no difference in expression levels as a result of my manipulations.

Has anyone had this scenario before? This is my first time doing these transfections and also the first time our lab has done these experiments so we're kind of new to it

Thanks

smr86



Why don't you transfect your cells and then, after 24 h, treat with IL-1B for 30 min and then harvest the cells?


Good idea thanks, I might just give that a try!!




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