Protein Removal from cell lysate - leaving RNA intact! (Jun/12/2009 )
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.
getting pure RNA without using solvents nor colums? how?
either to use kits(Ambion) or to use Triazol reagent (Invitrogen)