I am purifying 30s and 50s ribosome subunits to successively perform studies on their ri-association upon inhibitor addition.
I have never carried research on ribosome but I have been teaching molecular biology for a while, theory may help you a bit. Large and small sub-unit association-dissociation is driven by the presence of co-factors to promote association these are RNA and tRNA. For the big subunit to be efficiently recruited to the small subunit the small subunit must be bound to an mRNA. This mRNA has to be well positionned (with its RBS), the N-formyl-methionine-tRNA will position itself allowing association of the large subunit. Feed your reaction with RNA and appropriate tRNA and you will drive the reaction towards association.
Just dialyze your subunits against Hepes buffer (25mM of Hepes pH 8.0, 10mM of Mg acetate, 100mM of K acetate, 4mM of DTT). Carry out the procedure about 15 min and 30°C.
upd: I`m so sorry, but I made some erratum earlier. Just noticed. Now is fixed.
Original paper also added.
Thank you Hugo, Adam and Ivan for your answers.
@Ivan Sorokin: at your knowledge which is the optimal concentration of subunits to start with dialysis?
It`s not so important. But you need the equivalent concentrations of 30S and 50S in solution. Subunits should be in the "one to one" ratio.
Good luck!
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