I used nuclear extraction kit from Milipore 

Hi Ahmed,

Thanks for your reply. I need to solubilize the whole proteins including the nuclear proteins. So I am looking for the lysis buffer which could disrupt the nuclei. I think the nuclear extraction kit will just extract the nuclear protein separately.

Hi Xiaojing,

"Soluble" is a term that you will need to define for your particular project. There is no standard for solubilizing proteins because the biochemical profiles of proteins can be very different.  If you are doing a proteome screen, you can set different types of conditions and do sequential solubilizations to identify protein complements that are soluble under each tier.  If you are interested in a particular protein you can test for conditions that solublize your protein while still retaining it's activity.  That includes pH, salt concentration and type, and detergents.  There is no one size fits all; there is no "standard".  

A non-ionic detergent such as Tx100 will readily remove the nuclear envelope of all mammalian cells I've worked with, as long as you are well above the CMC of the detergent (approx 0.2 mM).  0.5% Tx100 (approx 8.5 mM based on avgMW of 625 and density of 1.07) typically does the trick for me.  You will also need to treat cells with DNaseI or benzonase to digest the DNA to release additional proteins and tame the viscous mess that results from lysing the nuclei.  If you are looking for a nuclear protein, you can enrich by lysing cells in the presence of a very small amount of Tx100 (0.025 - 0.05%) to help you lyse the cells, then recover nuclei by low speed spin (maybe 6000 rpm in microfuge) and lyse them separately in a somewhat higher concentration of Tx100 (0.25% would probably work well).

This will give you Tx100-soluble proteins. 

Hope that helps!

 Hi Becky,

Sorry for the long delay to reply. I just saw your helpful answer!!!! 

I tested two different lysis methods. Both are with the same lysis buffer, but in different mechanistic ways. 

After harvesting from the flask, cells were centrifuged at 120 g for 6 min to remove the media and PBS. Then flash freeze in liquid nitrogen. Cells were lysed with the lysis buffer (shown as below) (310 uL for T75 flask). First method: The cells were homogenized by forcing through a 27G syringe needle for 25 times (named Old). Second method: The cells were homogenized by forcing through a 27G syringe needle 25 times, followed by additional 31G syringe needle for 10 times (named New). After lysis, half of samples were centrifuged with 14,000 g for 15 min. The supernatant (S) and pellet (P) were carefully separated into two EP tubes. The total (T) protein, S and P were detected with WB for nucleoporin (nuclear envelope protein). I hope to see the same intensity of T and S, no intensity in the P. But the tricky thing is that I get the good result in the old lysis method (as you will see in the attachment pic), while I see nucleoporin in the pellet with the new lysis method! Could you please let me know how you test whether the nucleus membrane has been disrupted? Thanks a lot.

Lysis buffer: 20 mM Tris pH 8, 2 mM MgCl2, 1% (w/v) Triton X-100, Complete-EDTA-free protease inhibitor (Roche Diagnostics), 0.2% (w/v) sodium fluoride, 0.2% (w/v) sodium orthovanadate, 20 U/mL benzonase, 1 mM PMSF).