Mammalian cell lysis and fractionation

[robert clay]

Hi everyone,

I've been trying to isolate proteinaceous inclusions from cultured mammalian cells. To that end I need a way to lyse these cells without using a detergent (or perhaps a non-ionic one if absolutely necessary). I've tried mechanical stress but that tends to break open the nucleus as well (which is a problem since my protein of interest is also found in the nucleus).

Does anyone know a method of lysing mammalian cells that leaves the nuclei intact? Hopefully then I could pellet the nuclei whilst leaving any inclusions free with the cytosolic proteins.

Thanks!

[relaxin]

Most people lyse the cells with the nuclei intact by using 0.2% NP-40 in a low salt buffer.

[mchlbrmn]

If there's a french press somewhere about that you can borrow, you could consider that alternative. There's actually one in my hood that I used a few times for a different type of protein isolation from bacteria, but I read it can disrupt cells whist leaving the nucleus.

[dutchtech]

http://www.ncbi.nlm.nih.gov/pubmed/22134128

how about this?

[morkfromork]

You just want to do a nuclear/cytosolic fractionation. There are a number of classic protocols for this. Usually they involve plasma membrane lysis in a a low salt buffer (10mM KCl) by addition of a small amount of NP-40 or by mechanical disruption with a dounce homogenizer. The nuclei can then be pelleted out of the cytosol fraction. Nuclear proteins can then be extracted by addition of "high salt" buffer (~420mM NaCl). There are a number of variations on this protocol and I think there are even kits out there (although the protocol is dead easy using standard lab reagents).

[memari]

http://molecularbiology.forums.biotechniques.com/viewtopic.php?f=4&t=31718