Preparation of Cytosolic Extracts.
Specimens are snap-frozen in liquid nitrogen immediately after surgery and stored at –80 O C until extraction.
Frozen tissues (20 – 100 mg) are pulverized on dry ice to a fine powder and added to 10 volumes of extraction buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10 g/L of NP-40 surfactant, 1 mM phenylmethyl sulphonyl fluoride, 1 g/L of aprotinin, 1 g/L of leupeptin).
Suspensions are incubated on ice for 30 minutes, with repeated shaking and vortexing every 10 minutes.
Mixtures are then centrifuged at 14,000 rpm at 40 0 C for 30 minutes and the supernatant (cytosolic extract) is collected and stored at –80 0 C until further analysis. Protein concentration of the extracts is determined using the bicinchoninic acid bicinchoninic acid method, with albumin as standard (Pierce Chemical Co., Rockford , IL ).
The cytosol can then be used for numerous different analyses, by use in many protocols.