Isolate leukocytes from whole blood
Complement Pathway
1) For each 5 mL of blood, add 45 mL of room temperature 0.17 M Ammonium Chloride solution to lyse red blood cells. The cells will not correctly lyse if the solution is cold.
2) Incubate for 5 minutes on a rotator. Do not exceed 5 minutes or white blood cells begin to lyse.
3) Centrifuge for 5 minutes at 2000 RPM.
4) Aspirate supernatant, resuspend pellet in ~50 mL cold 1x PBS.
5) Centrifuge for 5 minutes at 2000 RPM.
6) Aspirate supernatant.
Cell lysate preparation
1) Add 1.0 ml of ice cold fresh RIPA buffer to the cell pellet
2) Gently pipet several times and let set for 15 minutes at 4° C on ice. This step will allow the lysis buffer to act on the cells and will increase the total yield of soluble protein.
3) Sonicate each sample on a 70% duty cycle or less by placing only the very tip of the pin into the vial, then slowly lowering it into the lysate until it foams completely and then stop. Alternatively, pass the lysate through a 21 gauge needle to shear the DNA & incubate 30–60 minutes on ice.
4) Microcentrifuge cell lysates at 12,000xg for 15 minutes at 4°C. 5) The supernatant fluid is the total cell lysate. Transfer the supernatant to a new microfuge tube and discard the pellet. Quantitate the protein amount by Bradford or BCA.