I am working on separating the soluble and insoluble proteins in cells. However, after the literature searching, I found there are many different lysis buffers, which means the corresponding soluble/insoluble proteins are different. Is there any standard for classifying the soluble and insoluble proteins? If there is, could anyone recommend me the lysis buffer strong enough to disrupt the nuclei, but not to solubilize the "insoluble" proteins? Thanks a lot!
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!
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