protein was still maitained in pellet after RIPA lysis? - in IP (Mar/20/2006 )

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?

what is the detergent in your RIPA buffer?
(you can find thousand different recipies)
what's the concentration?
you could maybe increase the concentration of the detergent?

Are you dealing with a membrane protein?

What kind of centrifugation protocols/speeds are you using?

-Matt

Thanks for the replies.

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

I centrifuge it in full speed by a few seconds.
Maybe I should try it in lower centrifuge?