*4.8. Cell Membrane Extraction*

Cell pellets harvested and resuspended in 20 mL of homogenization buffer (50-mM Tris, pH 7.4, 250-mM sucrose, and 0.25-mM CaCl2) containing protease inhibitors (1-mM pepstatin, 1.3-mM benzamidine, and 1.8-mM leupeptin). All the subsequent steps were done on ice. The cell suspension was then placed in the cell disruption vessel for nitrogen cavitation at 500 psi (Model 4639 Parr Cell Disruption Vessels (Parr®)). The vessel was incubated on ice for 15 min. Pressure was released slowly, and sample collected dropwise and centrifuged for 10 min at 560× *g* at 4 ◦C. The supernatant was collected, and the pellet, which contained unbroken cells and debris, was discarded. The supernatant was then ultracentrifuged at 100,000× *g* at 4 ◦C for 20 min. The supernatant was carefully discarded and the membrane pellet resuspended in 0.5–1-mL Tris sucrose buffer (TSB) (50-mM Tris-HCl, pH 7.4, and 0.25-mM sucrose). A needle syringe (0.45 mm × 13 mm) was used to resuspend the membrane proteins in TSB. Protein concentration was determined using a bicinchoninic acid kit (Pierce, ThermoFisher). Samples were kept on ice during the membrane preparations and were mixed with a sample buffer for loading on sodium dodecyl sulphate (SDS) gels.
