I have a floor centrifuge but it only goes up to 25,000 g. My old protocol requires an ultracentrifuge and 40,000 g, but it is non-functional. I am assuming I can spin at 25,000 g for longer time, but thought I would ask in this forum.
the usual way to isolate polysomes is by ultracentrifugation in a sucrose gradient. I've heard of protocols for separation of ribosome particles by FPLC but never tried them. Such an approach could work with polysomes as well. There is a kit supplied by Illumina for ribosome profiling studies, http://www.illumina.com/products/truseq-ribo-profile.html. As everything Illumina sells it is way overpriced, but an ultracentrifuge is much more expensive anyway.
I tried 35,000g for 2.5h before, and it worked. Although the profile did not look pretty, I can tell which is the polysome fraction..
Hey Anton, thanks for the suggested kit. Our ultra is dead and out for the count and I will investigate the kit you suggest!
Hi Jingping, Thanks for the info! I might give it a shot in our Sorvall centrifuge that goes up to 25,000 g.Or I might try to find an ultra that I can use. I have some friends at Texas A&M University.
Hi Felix,
So far the ultracentrifugation is required in order to isolate ribosomes. But there is an alternative strategy called TRAP and it's based on affinity purification (http://www.nature.com/nprot/journal/v9/n6/pdf/nprot.2014.085.pdf).
Hi Felix,
i hope you centrifuge is repaired now...
if you are interested my startup will release soon a new kit for active ribosomes isolation.
One step purification with mane tic beads.
But this version can be used only for RIbosome profiling experiments.
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