I am isolating proteins from crude nucleus using high salt concentration( 300-500 mM NaCl). Basically, I have to get nuclear proteins which will be used for CO-IP. In protocols I tried- a) 0.2mM EDTA along with high salt concentration (500mM releases DNA bound proteins readily).
b) High salt conc(300mM) + DNase 1(3mM MgCl2, without EDTA). - and I GOT nuclear proteins,using both of the protocols.
My question is, how does high salt conc cause nuclear membrane lysis, and how does 500mM NaCl extracts DNA bound proteins better than 300mM NaCl?
I would really appreciate your answers,
Thank you.
Hi Ashish. As far as I know, those salt concentrations do not cause nuclear membrane lysis. They do, however, allow you to extract a certain range of nuclear proteins, probably more so if you use DNAse. Bare in mind that a simple high salt nuclear extraction might not allow you to extract some or all of your proteins of interest. If you want to extract as much protein as possible for a Co-IP, it might be worth trying a buffer like RIPA (I wouldn't go harsher than this, as protein interactions would likely be affected too much).
Hi Ashish. As far as I know, those salt concentrations do not cause nuclear membrane lysis. They do, however, allow you to extract a certain range of nuclear proteins, probably more so if you use DNAse. Bare in mind that a simple high salt nuclear extraction might not allow you to extract some or all of your proteins of interest. If you want to extract as much protein as possible for a Co-IP, it might be worth trying a buffer like RIPA (I wouldn't go harsher than this, as protein interactions would likely be affected too much).
Thanks José, yes you are right about the efficient extraction of some of the nuclear proteins, precisely I need soluble and insoluble nuclear protein(Nucleolar) fractions. RIPA was in my mind but seems very harsh, especially with detergents. I do not want to treat my proteins harshly which might affect the interactions, because apart from my BAIT other interacting partners are not known to me, so can't predict how would they behave.
Thanks Nima for you comment on the above, you are right that it is not feasible to increase the salt concentration gradually, if you dealing with small scale.
However, I would really appreciate if you can suggest me any other protocol, milder one, for the nuclear protein isolation.
Extraction buffers contain no denaturing detergent (SDS) and proteins are extracted in native forms. Increasing NaCl to 0.7-0.8 M (final) results in histon extraction.
We have used FractionPrep Cell Fractionation Kit from Biovision succesfully. Good luck!
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