Buffer is filter sterilized and stored at 4°C. A 5X stock can be made, filtered and stored at 4°C for at least 6 months.
When ready to use add the following components fresh:
1 mM PMSF
5X PIN (Protease Inhibitor cocktail)
1 mM DTT
Note: You will require the use of the TL-100 Ultracentrifuge (Beckman) for at least 25 minutes. Both the 45 mL Nitrogen Bomb (Parr Instruments) and TLA-100 rotor should be pre-chilled at 4°C.
1. Using a rubber policeman scrape the cells from the plate into their media.
2. Place the media into a Falcon tube. Pellet the cells in a clinical centrifuge set on 3 (mcf-7) or 6 (rat-1) for 3 minutes.
3. Remove the supernatant (discard appropriately). Resuspend the cells in 5 mls of Cell buffer.
4. Pellet the cells again in the clinical centrifuge at the same speed and duration as above. Repeat this washing step.
5. Finally resuspend the cells in an equal volume of Cell buffer.
Varying the conditions for cavitation can yield different lysates. Here are the conditions for maintaining mitochondrial integrity.
1. Close the bottom (collection) valve on the nitrogen bomb. Put your cells into the chamber.
2. Push the rubber gasket on the pressure cap to the top. Slide the bomb onto the gasket and hand tighten the screw cap (do not over-tighten or rotate the chamber once attached).
3. Close the pressure control valve.
4. Open the main tank valve slightly (1/4 or less of a turn).
5. Gently open the pressure control valve until the desired pressure is obtained. [Under these buffer conditions, 150 psi is the highest pressure you can apply and still maintain the mitochondrial integrity]. Close the pressure control valve once the pressure is reached. Wait 15 minutes. (If you are using higher pressures you may notice a slight decrease in the chamber pressure over time, adjust accordingly).
6. During this incubation the bomb in surrounded by ice packs.
7. Place an Eppendorf tube (prechilled) at the base of the collection valve. Slowly open the collection valve to release the lysed cells.
8. If you have many samples rinse the bomb first with 70% Ethanol and then with Cell buffer before you begin your next sample.
1. Spin down the lysate in a micro-centrifuge for 2 minutes at 500 x g (this will pellet the unbroken cells and nuclei).
2. Gently remove the supernatant. This is the "Whole Cell Lysate".
3. Using the TLA-100 (Beckman) rotor mitochondria can be pelleted at 5 000 rpm (~1 000 x g) for 25 minutes at 4°C.
4. Light membranes can be pelleted at 51 000 rpm (~100 000 x g) for 60 minutes at 4°C .
Fractions were assayed via Immunoblots with anti-Hsp60, anti-Hsp27 and anti-cytochrome c antibodies.
The bomb should be rinsed thoroughly with 70% Ethanol ( DO NOT BLEACH ). It may be cleaned with a mild detergent if necessary.
Parafilm the valve and opening. Store the bomb in the cold room.