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 .
