Protein synthesis inhibition - (Jul/13/2009 )

I need an advice. I plan to treat MCF-7 cells with my test compound. First I need to stop nascent protein synthesis because I want to work only with proteins existing before treatment. For this purpose I want to use cycloheximide but I don't know what concentration I should use and how long incubate with cycloheximide before adding my test compound. Should I wash cells from medium containing cycloheximide before adding my compound? Maybe you recommend different method than this with cycloheximide. Thank you in advance for some information.

50-100 micromolar cycloheximide should be heaps, it is apparently effective within 15 minutes of addition, but it would probably be best if you waited for 30 min to one hour to ensure effectiveness. As far as I recall, CHX only stops about 70-80% of protein synthesis, and emetine is more effective, though CHX is more commonly used. Ideally you will do titrations of the amount and times to use CHX before deciding on what you will do for your experiments.

AS to whether you should wash your cells after removal of CHX, yes, you don't want residue interfering with your experiment.

First of all thank you for your helpful advice. I've got just one concern. 50-100 micromoral of cycloheximide seems to me it's too much. Isn't it 5-10 micromoral, possibly?

Regards

lukasz

bob1 on Jul 13 2009, 05:47 PM said:

Sorry, I was mis-remembering my experiments. I meant 50-100 ug/ml, though the attached document suggests 1-100 ug/ml

Thank you a lot. Cheers
http://www.protocol-online.org/forums/styl...fault/smile.gif

lukasz

bob1 on Jul 15 2009, 05:43 PM said: