Crude Membrane protein extraction from tissues
Thaw 20–50 mg of tissue or 15CM dish cells in 1 mL of high salt buffer.
Homogenize tissues using an IKA Ultra Turbax blender at maximum speed ( ∼25,000rpm) for 30 s. 
Ultracentrifuge the suspension in Beckman MLA 130 at 100,000 g for 10 min.
The tubes should be balanced to a difference less than 50 mg.
Discard the supernatant and homogenize pellets in 1 mL of carbonate buffer as in step 2 .
Incubate for 30 min with gentle mixing.
Collect the non-soluble material by centrifugation as in step 3 (If the content of integral membrane proteins in the purified membranes is not at least 30–40% of total identified proteins, steps 4–6 can be repeated two to three times).
Discard supernatant and resuspend pellets in wash buffer as in step 2 .
Collect the crude membranes by centrifugation as in step 3 .
