I would like to find cell lysis method to solubilize/break-up nuclear membrane without breaking/denaturing protein-protein interaction of protein complex in nucleus before doing CO-IP. I need to find method to breakup nuclear membrane, however It seems that RIPA buffer and sonication method are not recommend which can denature protein-protein interaction. Could you recommend me a method to break up nuclear membrane to get "intact" nuclear protein complex for CO-IP? especially chemical method.
Thank you so much
Hello
find the attachment & link here , i hope help you
Good Luck
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693241/
Hi Asa, I have a protocol to isolate nuclear protein for gel shift assay. I think it should be ok for you to use.
A slight modification of the method of Dignam et al. (Nucleic Acids Research, Volume 11, p1475-1489, 1983) is as follows. Cells were washed in phosphate-buffered saline (PBS) and resuspended in hypotonic buffer (HB; 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HIEPES; pH 7.9 at 4°C],
0.75 mM spermidine, 0.15 mM spermine, 0.1 mMEDTA, 0.1 mM EGTA, 1 mM dithiothreitol, and 10 mMKCl) and allowed to swell on ice for 10 min. Cells were pelleted and resuspended in 2 packed-cell volumes of HB before
homogenizing [10 storkes of a Kontes all glass Dounce homogenlzer (B type pestle)]. After homogenization, 0.1 volume of sucrose restore buffer (1 part lOx HB, 9 parts 75% [wt/vol] sucrose) was added, and nuclei were pelleted at 16,000 x g for 30s. Cytoplasmic proteins were retrieved as the supernatant from the nuclear pellet. Nuclear proteins were extracted with buffer C [20 mM HEPES (pH 7.9), 25% (v/v) glycerol, 0.4 2 M NaCl, 1.5 mM MgCl , 0.2 mM EDTA, 0.5 mM PMSF and 0.5 mM DTT] and processed by the method of Dignam et al. Extracts were prepared in the presence of the protease inhibitors phenylmethylsulfonyl fluoride (0.25mM) and aprotinin (5 ,ug/ml). Good luck!
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