Hello Linda,

I have used a sucrose gradient ranging from 15% to 60% in order to isolate mRNA from the polysome and the monosome fractions for several experiments with HeLa cells. I find that this concentration range gives a good resolution of the individual ribosome peaks (small/large subunits, monosome, some disomes and polysomes are all clearly visible) and can still be handled well, because it's not too viscous. Concerning the pmol amount of loading I cannot give you a definitive answer. Do you mean the maximum amount to prevent smearing of the peaks or the minimum amount? I experienced that HeLa cells grown to 70%-90% confluency in a 15cm cell culture dish give good results (should be around 10 million cells). I would try to prevent higher confluencies because this can lead to translational contact inhibition. Obviously, the quality of the gradient is important for a nice seperation of the peaks. I had very good experiences with a machine called "Biocomp Gradient Master" to generate very clean gradients. Also I found it very useful to use a peristaltic pump that pushes the gradient out of the tube which is pierced with a needle in the bottom through which 70% sucrose is flowing. Concerning the centrifugation: I used a SW40 rotor with Polypropylene tubes (14 × 95 mm) with a usable volume of 12ml. With the SW40 rotor I ultracentrifuged the tubes for 3h at 4°C and 39,000 rpm.

I also attached the detailed protocol (PDF format) that I and some others at my institute used for sucessful experiments with human cells and C. elegans cells. In the protocol I'm citing a paper by "Aeschiman et al., Methods, 2015" which gives many details about ribosome purification which go beyond just running a gradient.

Maybe this will help you to get a better overview!

Best,

Johannes

Hi Johannes,

thanks a lot for your answer. I mean the maximum amount you can load on a gradient to prevent the smearing of peaks, but also to provide clean ribosomes. Last time I tried 10-40% and 20-50% and the gradient profile looks quite good. However, if I put the fractions on a gel there is still a lot of dirt inside. I purify the ribosomes via a His-trap before I load them on a gel, but unfortunately they were still not very pure after the gradients. So I hope this can be improved by a different gradient or by loading less on a gradient. 

Best,

Linda

Hi Chad,

thanks a lot for your answer. I actually want to avoid to pellet the ribosome, since I am afraid I will lose a lot and probably destroy it. I increased the salt concentration for the gradient and now it seems to work fine. However, I can not separate 50S from 70S with my gradient. Is this possible with the sucrose cushion?

Best,

Linda

Thanks a lot Chad. This is very useful information.