• 60 mL ELISA protein extraction buffer solution
• 1 mL 100 X Broad Spectrum Phosphatase Inibitor (store at -20 °C)

This ELISA protein extraction buffer contains a mild detergent enabling desolution of cell membranes, solubilization of most membrane proteins and suspension of cytosolic proteins into the buffer. A separate vial containing a 100X broad spectrum phosphatse inhibitor cocktail is provided to preserve seryl/threonyl and tyrosyl phosphorlation when needed.

1. Prepare 500 μl ELISA protein extraction buffer for each 10-cm dish, or 200 μl for each well in a 6-well dish. Make the solution ice cold and add protease inhibitors, including serine protease inhibitor (such as PMSF). If needed, also aliquot provided phosphatase inhibitor which is provided 100X.
   2. Remove media and rinse cells three times with PBS.
   3. Add 500 μl ELISA protein extraction buffer into each 10-cm dish. Rotate the dish to cover cells with ELISA protein extraction buffer and place on a bed of ice for 5 min.
   4. Scrape cells off the dish, and transfer to an appropriate tube. Rotate and tap the tube several times to solubilize membranes. You should observe fibrous materials which correspond to genomic DNA, an indicator of cell lysis. If fibrous materials are not apparent, add 20 μl aliquots of ELISA protein extraction buffer, and rotate and tap until the fibrous materials are observed. Keep the tube on ice for another 15 min.
   5. Centrifuge the tube at 4oC for 15min and collect the supernatant, which will be used in the ELISA assay. A protein concentration assay, such as the Bradford Assay, can be used to quantify total protein concentration. The supernatant can also be stored at -80oC long term.