To measure protein half-lives in cultured mammalian cells (HEK293), we used cycloheximide at a concentration of 100 mkg/ml. The control protein which is GFP fused to the C-terminal degron of ornithine decarboxylase (ODC) decays slowly with a half-life of 2-4 hours. Under these conditions the level of a test fusion protein does not decrease. However, other experiments suggest that the test protein should degrade with the same rate as ODC. I think that 2-4 hours is a very long time for the half-life of ODC. Is the 100 mkg/ml of cycloheximide sufficient to stop translation? Should we increase its concentration? Are there alternative methods to stop protein synthesis in mammalian cells?
Pseudomonas exotoxin A, and Diphtheria toxin, shut off protein synthesis very rapidly. Both of them ADP-Ribosylate the elongation factor 2 protein in a catalytic reaction, so as little as a single molecule of the toxin can stop all protein synthesis in a cell. These toxins, like cycloheximide, only stop cytoplasmic protein synthesis, so proteins synthesized in the mitochondria can still be produced.
Yes there are alternative methods, you can also use harringtonine which block ribosome at the AUG codon . However ODC is strongly regulated by polyamines at translational and post-translational levels. So this could lead to misinterpretation if somehow you stabilise ODC in presence of CHX.
I think using siRNA against the RNA that codes for your protein may be a very easy way since transfection is very easy and siRNA is very specify, so you would degrade the RNA and the protein would never be made, and you can re-express the protein by not using the siRNA for sometime, a useful method to shut off protein synthesis transiently.
I agree with Ivan. All of the serious biochemists I've consulted with have preferred cycloheximide.
You might (but probably don't) need a higher concentration, but you should probably verify that you've actually blocked protein synthesis by using a labeled amino acid (either radioactive or through BONCAT/Click). Anisomycin is another widely-used alternative (and often used as the control to make sure any cycloheximide effect isn't off-target), but make sure the cycloheximide is working first. If everything is working, trying moving your GFP to another part of the protein---perhaps you've disrupted a degradation signal.
Hi everyone! I want to use cyclohexamide (CHX) in HEK cells, do you think that I should use a DMEM with or without FSB? Some authors use FSB and some others don't. What would you recommend? 100 uM of CHX would be enough right? I would like to take samples 4, 8 and 12h after CHX treatment.
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