membrane protein extraction buffer - (Oct/14/2009 )

Hi!
Could anybody give me an advice which lysis buffer to use for membrane protein extraction from culture cells. I want to do protein electrophoresis (with denaturated proteins) and I have expressed my protein in COS cells, the protein itself is ATP/ATP carrier, which is transmembrane mitochondrial protein. I have looked for several protocols and I am not quite sure which method would be the best. I have read that some people suggest using the RIPA buffer, but at the same time some people say that its not good for membrane proteins.
Thank you very much in advance,
Minna

pokemon on Oct 14 2009, 09:23 AM said:

Hi Minna,
I use this protocol to determine the translocation of PKCd to the membrane in hepatocyte cultures. Note that it is Triton-soluble membranes. Hope it helps you.

Homogenization Buffer TritonX Buffer
10ml 1.0ml
1M Tris pH 7.4 100ul 1M Tris pH 7.4 20ul
100X Protease Inhibitors 100ul 0.5M NaCl 4ul
1M Na Vanadate 10ul 20% Triton X-100 50ul
100uM Okadaic acid 10ul 100X Protease Inhibitors 10ul
Sucrose (Do this first 0.873g Water 0.916ml
because it swells the volume)
Take to 10ml with Milli-Q H2O

Place culture dish on ice. Rinse twice with cold PBS.
Add ~125ul H Buffer to 6cm culture dish on ice. Scrape into microfuge tube.
Freeze -80 C for 15minutes.
Thaw and then pass through 25g syringe 7X.
Syringe the 7th (1.0ml, MAX capacity) into Beckman thick walled centrifuge tube (343778) tube. Spin at 53000 rpm, TLA120.2 rotor 4C, 30 min
(100,000g)
Transfer to tube marked CYTOSOL


Add 40ul Triton-X Buffer to each pellet and gently resuspend.
Incubate on ice 30min.

Centrifuge at 53000, TLA 120.2 rotor 4C, 30 min

Transfer supernatant to tube marked Triton-soluble MEMBRANE.