I am just starting polysome fractionation (mammalian cell lines). I have an extra peak, could be 43S (40S + Initiation factors), but wanted to test the fractions for subunits specific to 40S and 60S. Does anyone know any known markers used to test the fractions for each subunit? (Either by western blot/ribosomal proteins or RNA analysis/rRNAs).
Thanks.
Here is a peak pattern that might help.
http://www.biomed-search.com/search?q=monosomes&s=0&r=9
Is there an additional peak in between?
If you just need to identifiy which subunit is present in your fraction, looking for the size of rRNA would be easiest. Extract RNA from your fraction and run it on an agarose gel together with RNA from the whole prep (control), stain with any appropriate stain like Sybr Green II or ethidium bromide. The 5S,18S and 28S rRNA will appear as prominent bands in the control and you should be able to confirm that your 43S fraction does correspond to the small ribosome subunit
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