Mem per plus membrane protein extraction kit

The isolation of membrane-associated proteins was performed using the Mem-PER™Plus Membrane Protein Extraction Kit (Invitrogen) (32 (link)). In summary, cells were harvested from suspension cell cultures by centrifugation at 300× g for 5 min. The obtained cell pellets were washed with 3 mL Cell Wash Solution and centrifuged at 300× g for 5 min. After the supernatants were removed, the cell pellets were resuspended in 1.5 mL Cell Wash Solution, transferred to a new tube, and centrifuged at 300× g for 5 min to discard the supernatants. The resulting cell pellets were mixed with 0.75 mL permeabilization buffer to obtain a homogeneous cell suspension and incubated for 10 min at 4 °C. The permeabilized cells were centrifuged at 16,000× g for 15 min, and the resultant supernatants containing cytosolic proteins were removed and transferred to a new tube for detection. The pellets were resuspended in 0.5 mL solubilization buffer, and the mixtures were incubated at 4 °C with constant mixing for 30 min. The resultant solution in the tubes was centrifuged at 16,000× g at 4 °C for 15 min. The supernatants containing the solubilized membrane and membrane-associated proteins were moved to a new tube for analysis. The proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and detected with various antibodies during western blotting.