protein was still maitained in pellet after RIPA lysis - IP (Mar/19/2006 )

both lysate suspension and pellet samples are checked by western. Then I found the protein still mainly exist in cell pellet after RIPA lysis and high speed centrifuge. (other proteins are ok, means no problem from RIPA buffer.)

Except for sonication after adding RIPA, any other suggestions?

You probably thought of this but you could try a stronger detergent. RIPA uses Triton X I think right? You might try SDS at a couple different concentrations and see if you can solubilize the protein. Also If you know in what compartment it normally is inside the cell you can maybe "steal" a protocol for solubilization of X compartment or something from the literature. For instance if it's always in the lipid rafts or something that might explain your problem


Good Luck

Many thanks for your kind suggestions!
My protein is in nucleolus. To my known, it's not a lipid or membrane binding protein.
Maybe you are right, I would try higher concentration of SDS and lower centrifuge speed.
What I used in RIPA is already NP40 with SDS:


the RIPA I used is:

1% NP40
1% Na-deoxycholate
0.1% SDS
150mM NaCl
10mM Na-phosphate, pH7.2
2mM EDTA
50mM NaF
5mM beta-glycerolphosphate

fresh add
4mM Na3VO4
1mM DTT
1mM PMSF
20ug/ul aprotinin
100 U/ml benzonase

30min 4C, shaking

Hello,

You may pick a protocol for EMSA (electrophoretic migration shift assay) to extract nuclear proteins.
I know that TritonX-100 will not extract nuclear proteins for example (I don't know for NP-40, but might be the same).