How are protein extracts prepared?
Frozen tissue is minced and homogenized in cold modified RIPA buffer [PBS (pH 7.4), 0.25% Na deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, 1 mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml Leupeptin, 1 µg/ml Pepstatin A). Based on numerous mammalian tissue isolation protocols reported in the scientific literature and protocol manuals, we decided to use the average, recommended 1:9 (w/v) ratio of tissue to protein extraction buffer for maximize overall protein recovery. The first step involves mechanical shearing with a Polytron-type homogenizer at 35,000 rpm until the tissue is dispersed. The tissue solution is further disrupted by liquid shearing in a Dounce or Potter-Elvehjem homogenizer. This solution is centrifuged (20,000 x g; 20′; 4 degrees C) which is adequate to pellet most insoluble proteins, extra-cellular matrix, any intact nuclei, lysosomes and mitochondria. The supernatatant, which contains soluble proteins (cytosolic, nuclear and membrane) proteins, ER membranes and ribos
