RIPA buffer - problems (Oct/10/2008 )

I'm using a normal described RIPA buffer for protein extraction. See recipie:

5 ml 1M tris-cl PH 7.4
30 ml 5 M NaCl
5 ml 20% NP-40 or ( I used TRTITON-X since NP-40 is not comercially available anymore)
5 ml 10 % sodium deoxycholate
0.5 ml 20% SDS
50 mL ddH2O

the problem is that the pellet does not stick to the ependorf after centrifugation (10 min max speed..and even twice)...it slices dwon..or if I try to pippet the supernatant I can see a smear coming out of the pellet.....

What's wrong?

thanks

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carry out all steps on ice....keep on ice....the sticky clump is the genomic DNA...you can use DNase to eliminate that but why not simply keep on ice?

the best RIPA recipe is:

- Tris 50mM
- Nacl 150 mM
- SDS 0.1 %
- Na.Deoxycholate 0.5 %
- Triton X 100 or NP40 1%
- 1mM PMSF
- Roche Complete Protease inhibitor tablet or PIERCE Halt inhibitors


Do Not put more than 1% NP40 or Triton X-100

RIPA for 50 ml
- 0.302 g Tris  pH 7.8
- 0.438 g NaCl
- 0.05 g SDS
- 0.25 g NaDeoxycholate
- 0.5 ml Triton X 100 or NP40
- 0.0087 g PMSF
- Roche Tablet according to manufacturer's guide

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