Cellular-nuclear protein isolation by freeze-thaw method
Hello everyone.. I have a query whether cell lysis by freeze-thaw method is good enough to break the nuclear membrane as well,while lysing the cells. I have to isolate the nuclear protein separately.
Thank you..
Certainly there is no general answer possible as this depends on the type of cells used and other factors. Commercial kits are around. See below one exemplary description:
Resuspend cell pellet in a cytoplasmic lysis buffer for 15 minutes. Add detergent and pellet the nuclei. Transfer the supernatant containing the cytoplasmic fraction and further isolate the proteins with cytoplasmic extraction buffer. After the nuclei have been washed, the pellet is resuspended in nuclei lysis buffer for 15 minutes. The nuclei debris is pelleted and the supernatant contains the nuclei protein fraction.
Thanks Arne.I have been trying this as you said. For cytoplasmic lysis I am using Tris+Mgcl2+NP-40+Protease-inhibitor,incubation then centrifugation finally getting the nuclear pellet, this pellet is washed with PBS and now re-suspended in Tris+LiCl+Glyecerol+Protease-inhibitor,ultracentrifugation.
Good I am getting the nuclear protein.
Now my main concern is that I have to pull-down the nuclear protein complexes,though the metod I am using is not that rigrous,but it could hamper the protein-protein interaction. Thats why I was thinking to try freeze-thaw. Any suggestion abot freeze thaw or any other metoh that could be applied here.
Hi Ashu,
I cannot comment on freeze/thaw due to a lack of experience but separation of nuclei sounds good. The nuclear protein extract should be in the supernatant after centrifugation at about 20,000 g. Try these proteins, maybe it’s already quite well.