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Cycloheximide: positive control
Posted by: mb80 (IP Hidden, Junior member, 12)
Date: June 5, 2006 09:57PM

Hi,

I am trying to do a cycloheximide experiment to prove
that a particular gene is a direct transcriptional
target of a pathway. After blocking protein synthesis,
if I still see induction of gene "X" after activation
of my pathway of interest, I can say it's direct.

The problem is that I need a POSITIVE CONTROL to show
that I have indeed blocked protein synthesis.

The basic methods I have found all involve the measurement
of incorporation of labeled amino acids:

1) S35-methionine
2) C14-leucine
3) H3-leucine


I am trying to find the fastest, easiest, but also safest
METHOD for obtaining a positive control. I've heard S35
can be messy and a pain to deal with due to aerosols,
as I will need to do cell culture for this expt.
We currently do not have a dedicated incubator for
such plates, etc....

Does anyone know about C14-leucine or H3-leucine for
these types of experiments? I've found some old protocols
in older papers, but any words of advice would be helpful.

I've also thought about trying to find an "INDIRECT TARGET"
of my pathway of interest, and showing that such a target
FAILS to be induced after pathway activation in the presence
of CHX, but there is not an obvious example that comes to mind,
and the above approaches are probably more rigorous and better
anyway.


Thanks a lot for any input!!!
M

 

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Re: Cycloheximide: positive control
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: June 12, 2006 11:55AM

S35 is most often used for autoradiographical detection of protein synthesis. Yes it is some kind "messy" especially if you are working in a big lab. You can put activated carbon or charcoal canister in the incubator to absorb aerosol.

 

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Re: Cycloheximide: positive control
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 1, 2006 02:07PM

You can also purchase sterile charcoal filters that will fit into the lids of many culture dishes and plates to absorb the aerosol. Scienceware is one source of these filters.

 

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