Cycloheximide prevents run-off, so you should not use it here, except perhaps in parallel samples as a control.

cycloheximide should not bring ribosomes back to mRNAs. It traps elongating ribosomes by binding in the E site

As you can see in conditions  for measuring ribosome half-transit time, detailed in he attached paper, Cyclohexamide should not be used.

Hi,

Look at this paper: Inhibition of Eukaryotic Translation Elongation by Cycloheximide and Lactimidomycin, Nat Chem Biol. 2010 Mar; 6(3): 209–217. 

My feeling is that Cycloheximide would not allow puromycin to bind at the A-site because is affecting the E-site. You might try to use antibiotic inhibitors of translation that interact at the ribosome's small subunit. Usually, those interacting at the big subunit would affect puromycin activity, but those interacting in the small subunit would not (e.g. aminoglycosides or tetracycline). Unfortunately, this antibiotics might produce aberrant results in your ribosome profiling data. You have to test several of them before considering one. Another option is to add puromycin to the cells (to erase translating ribosomes) and later use cycloheximide to obtain your final arrested ribosomes.

Puromycin removes protein in the course of synthesis of the ribosome by substituting itself for the tRNA loaded with aa. Unfortunatly I do not know what becomes the messenger RNA.

As stated by all before me - cycloheximide should not be added as it blocks translocation. Without translocation puromycin will not be incorporated in the elongating polypeptide.

Cycloheximide should not be used in your case, as it prevents puromycin to work (and thus prevents run-off)

Just treatment with Puromycin does not release empty ribosomes from the mRNA (usually high KCl is needed for the dissociation or run off should happen). Cyclohexamide will hamper this process. Hence, as yourself observed, cyclohexamide will keep the polysomes intact.

You should not add chx in this experiment..it prevents run off.. the whole premise of your experiment is ruined... 

No CYH for this experiment. You want the ribosomes to "run off" the mRNA, not be stuck there

You should definitely not add cycloheximide at the same time as puromycin.  However, if you are treating solely with puromycin to induce ribosome release from mRNA, then post-treatment you would need a way of trapping everything that has not been released- otherwise run-off will occur during sample preparation due to lack of nutrients or other stresses. Cycloheximide could therefore be used post-treatment to achieve this- alternatively you could use formaldehyde to x-link any remaining ribosomes to the mRNA then run gradients

Thank you for all of your responses. I'm glad to find out that most of what you said are what I understand. I understand that concomitant treatment of puromycin and cycloheximide will prevent elongating ribosomes from running off (I actually found a paper nicely showing that). But in my experiment what I did was a "subsequent" treatment. Puro first, then CHX after.

Mark P Ashe's comment pointed out what I wanted to do. The reason I used CHX is to stabilize ribosomes on mRNA during sample preparation ("crosslinking" kind of idea).

Specifically, I treated cells solely with puromycin for 0,5, or 20min to induce ribosome release first. Then add in CHX and incubate for 5min in order to trap the ribosomes that have not been released during puro treatment. From that on I included CHX in all steps throughout sample preparation and sucrose gradient centrifugation to prevent any undesired dissociation. 

I'm attaching the chromatograph I obtained. They are WT (blue curves) and Ras-transformed (red curves) Mefs with no treatment or treated with puromycin (50ug/ml) for 5 or 20 minutes. CHX was applied to all of the 6 samples AFTER puro treatment. (The 80S peak got cut off at the top is due to the stylus going to the top of the chart paper and can't go higher.) I have no idea why the polysome peaks didn't diminish at all.

Any suggestions for me?

Thank you again! Zhizhou

If you add its only purpose will be"an added cost". If you are careful, then there is no need to even talk of cyclohexamide.

I have to confess that I don't understand- the blue profile looks exactly as you would expect- ribosomal material has migrated from polysome to monosome areas after puromycin treatment?  Or I may be missing the point entirely. Ras looks to be a different story.  Are you sure the puromycin is getting in and end labelling the proteins - you can test this on a Western (see Figure S6 Lui et al 2014 Cell Reports 9:944)

Mark P Ashe, thanks for your reply. I agree that the blue profile's monosome peaks are significantly bigger (which I would expect), but I'm concerned that the polysome peaks didn't get diminished at all. When I further did qRT-PCR for the fractions against Actin (serving as a positive control), its distribution didn't change between no puro and puro-treated conditions, again suggesting this experiment may not be working. Like you said, I'm not sure if puromycin got in or not. Thanks for the paper reference. It's a good idea. Also I'm going to try longer timecourse next time.