Hi Roberto,

Option Nr. 1 would be to use a different (more long lasting or stable) approach to get rid of your gene of interest, something like CRISPR. This would then allow you to grow as many cells as you need for polysome profiling. However, there are probably good reasons why you don't want to do that.

Option Nr. 2 would be the following, but I have to say that I have never tried it myself: Next to high quality input lysates, the most critical part of a sucessful polysome profiling experiment is the choice of 'steepness' your sucrose gradient (i.e. 20%-70% versus for example 60%-80% or 20%-50%). Such different gradients allow you to have a different dynamic range and resolution of your fractions. If you want to purify mainly polysomes you will need a different gradient compared to when you want to purify only the small ribosomal subunit. Since you are not interested in polysomes, but the ratios of 80S/40S/60S you will need a gradient that resolves well in the "low" molecular weight range, for example 20%-50% sucrose. That way, all polysomes that you are not interested in, will be stuck in the last fraction and they don't annoy you. But maybe you knew all this already. The important point is this: For your exact cell type you will have to find the best gradient. Therefore, I would propose to perform this experiment with a sufficient amount of you cells (but importantly no knockdown) and find the best gradient for you. Try to aim for as much 40S, 60S and 80S in single, but different fractions as possible.When you have found a good gradient for your cell type and biological question (high resolution of 80S/40S/60S ), I would suggest to use exactly this gradient for a smaller amount of your knock-down cells (use as much as you can possibly get without spending your entire budget on siRNAs...). Depending on the sensitivity of your hardware (absorbance measurement etc.) and final amount of cells, you might of course not see peaks. However, from your earlier characterization of the non-KD cells you already know exactly which fraction contains the 40S, which one the 60S and which one the 80S. To visualize this information and to semi-quantitativly analyze the ratios, you should then perform a Western blot against exclusive 40S and 60S marker proteins. You could use for example an antibody against RPS27 (MW: 9.5 kDa) to detect the 40S and an antibody against RPL9 (MW: 22 kDa) to detect the 60S. The 80S will of course contain both proteins. A Western blot containing all your fractions probed against both 40S- and 60S-specific antibodies should then give you the answer how the 80S/40S/60S ration changes with and without knock-down (re-do the experiment also with the exact same amount of cells for the non-knock-down condition to better compare the relative ratios).

Important: If you want to isolate RNA for sequencing this will not work, since quantities will be too low, but for Western this should not be a problem due to the high sensitivity.

I hope this makes sense. Good luck!

Johannes

Thank you Johannes!

Thank you (very in late...😅) for your kind reply!

(Sorry, I have lost your reply... 😅)

I will try what you recommend!

Bye!