2X Lysis Buffer (Item J)

2X Lysis Buffer should be diluted 2-fold with deionized or distilled water. Supplementation with protease or phosphatase inhibitors is recommended. You may use RayBiotech Protease Inhibitor Cocktail (AA-PI), RayBiotech Phosphatase Inhibitor Cocktail Set I (AA-PHI-I) or Phosphatase Inhibitor Cocktail Set II (AA-PHI-II), or the RayBiotech Phosphatase / Protease Inhibitor Set (AA-PIPHI). Alternatively, you can purchase inhibitor cocktails from another manufacturer and follow their specifications.

Rinse cells with PBS, taking care to remove any remaining PBS before detaching cells. Detach 2 x 10⁷ cells by trypsinization or scraping and transfer the cells to a microfuge tube. Centrifuge to pellet the cells and then remove any remaining buffer. Add ~500 µl prepared lysis buffer and pipette up and down to resuspend the pellet. Incubate the lysates with shaking at 4° C for 30 minutes. Spin down the tubes in a microfuge at top speed for 10 minutes at (4°C), and transfer the supernatants to a clean tube.

Transfer approximately 100 mg crude tissue into a tube with 1 ml 1X Lysis Buffer. Homogenize the tissue according to homogenizer manufacturer instructions. Transfer extracts to microfuge tubes and centrifuge at top speed for 20 minutes (4°C).