We tried to make cycloheximide chase assay in yeasts and found that even 200 mkg/ml concentration of cycloheximide in complete selective media does not stop our protein translation which is very unstable protein in vivo. Could you suggest an efficient protocol for translation inhibition in yeasts using cycloheximide or may be other chemicals and conditions (additives or media composition) that enhance their action? Could the failure with cycloheximide be a consequence of strain resistance to this drug?
The half-life of my protein should be less then 5 minutes, according to the published literature. We fused it to the reporter protein - beta-galactosidase. In earlier experiments half-life of this chimeric protein extended to about 30 minutes. However, now we could not even reproduce our old results - the protein is stable. We measure half-life by activity of LacZ. We think that one matter is in the composition of media.
As stated above, it is unlikely that your strain is resistant to CHX, but if you are worried you can easily test for growth in the presence of CHX. I would like to suggest a trivial explanation that you should rule out, is your CHX stock good?
We checked the strain and it is sensitive to CHX. The reagent seems to be OK. It is possible that my protein may be involved in anti-CHX response, and we think if the induction of its transcription can attenuate the CHX effect. We managed to work CHX at concentration 600 mkg/ml as it was suggested in the protocol of Thorsten Pfirrmann.
Tobias, we have never seen an effect of nitrogen source on the effectiveness of cycloheximide. Can you elaborate on other drugs whose effectiveness is decreased by ammonium suflate, I'm aware of G418 but not many others. Cheers
Thank you all for yours information. I just wondering that in case of I use a large amount of cell e.g 40 OD600 units, should I increase the concentration of CHX and how much would be the best?
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