ELISA Lysis and Protein Extraction Buffer

The 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.

Prepare 500 ul ELISA protein extraction buffer for each 10-cm dish, or 200 ul 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 add general phosphatase inhibitor.
Remove media and rinse cells three times with PBS.
Add 500 ul 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.
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 ul 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.
Centrifuge the tube at 4C 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 -80C long term.