Protocol:

1. Bring both Reagents to room temperature prior to use. If any precipitates are observed, warm the vial using a water bath until the precipitates have dissolved and then allow to stand on ice. 
2. In two seperate tubes, take the required volumes of Reagent A and Reagent B. Immediately before use, add PMSF to a final concentration of 1 mM to prepare Reagent A Working Solution and Reagent B Working Solution respectively.
3. Preparation of cell and tissue samples:
   • Adherent cells: Culture around 2 × 10⁷ to 5 × 10⁷cells, wash with PBS or treat with EDTA and mix thoroughly using a micropipette. Centrifuge for a few minutes to precipitate the cells. Discard the supernatant and collect the pellet. It is recommended to not to digest the cells with pancreatin to avoid degradation of protein.
   • Suspension cells: Culture around 2 × 10⁷ to 5 × 10⁷cells and centrifuge for few minutes to precipitate the cells. Discard the supernatant and collect the pellet.
   • Washing cells: Resuspend the cell pellet with ice-cold PBS, take a small number of cells for counting and then centrifuge the remaining cells at 600 × g at 4°C for 5 min to precipitate the cells. Discard the supernatant, then centrifuge the pellet at 600 × g at 4°C for 1 min to further precipitate the cells. Discard the supernatant completely.
   • Pretreatment of cells: Suspend 2 × 10⁷ to 5 × 10⁷cells in 1 ml of Reagent A Working Solution and incubate on ice for 10-15 mins.
   • Tissue samples: Homogenize 0.1 g of sample in 1 ml of Reagent A Working Solution and incubate on ice for 10-15 mins.
4. Transfer the cell suspension or tissue homogenate to a pre-cooled homogenizer and homegenize 30-50 times. Ensure that the cells are completely homogenised by performing a nuclei ring observation onto a glass coverslip. Repeat 10-30 times if homogenisation is incomplete. Avoid excessive homogenization as this may damage the mitochondrial membrane and may trigger the release of mitochondrial components.
5. Centrifuge the homogenate at 700 × g at 4°C for 10 mins to remove nuclei and unbroken cells. Carefully transfer the supernatant containing the cytosol protein to a new tube without disturbing the precipitate and analyze immediately or store at -70°C.
6. Centrifuge the precipitate at 14,000 × g at 4°C for 30 mins to precipitate the cell membrane fragment. Centrifuge again at 14,000 × g at 4°C for 10 sec and discard the supernatant. Add 200 µl of Reagent B Working Solution, suspend the pellet using a vortex mixer and incubate on ice for 5-10 mins. Repeat this step for 2 more times to extract the protein completely and centrifuge at 14,000 × g at 4°C for 5 mins. Transfer the supernatant containing the membrane protein to a new tube, analyze immediately or store at -70°C.

Notes:

• PMSF is not provided in the kit.
• The extracted protein can be assayed using Abbexa's BCA Protein Assay kits - abx090640, abx090642