Protein Removal from cell lysate - leaving RNA intact! (Jun/12/2009 )

hi,

I am trying to figure out how to remove proteins from a cell lysate (large cell number), the idea being to leave mRNA intact.

so, standard protocol involves the lysis and removal of nucleii. thats fine- making the cytosol extract. what i need to achieve is a near zero concentration of SDS at the end fo the procedure, with as much protein gone as possible.

obviously, i can go ahead and SDS precipitate proteins, then dilute the samples. that may work fine for what i need after (NOT PCR). i was wondering if there is an alternative that would leave mRNA intact, WITHOUT USING SOLVENTS!

this is an odd question i realize, i cant go into more info due to the nature of this work but its essential that i can get a pretty pure RNA prep, that is not column based and does not use solvents- i need a total RNA that is treated only to remove proteins from the lysate, nothing else.

I may just go ahead and dialyse the samples, post-precipitation, but that can lead to problems with RNA. if i can remove the proteins some other way. just a reminder, i will be using a cytosolic lysate so no issues with genomic DNA.

also digestions of proteins enzymatically are not what im after either. need to get them out, without fractionation.



anyone?

J

getting pure RNA without using solvents nor colums? how?
either to use kits(Ambion) or to use Triazol reagent (Invitrogen)