You should have the following stock solutions prepared:
250 mM sucrose stock solution
250 mL
sucrose
21.39 g
2 mM MgC2l stock solution
250 mL
MgCl
0.0476 g
20 mM HEPES pH 7.5 stock solution
250 mL
HEPES pH 7.5
1.192 g
1 mM EDTA stock solution
250 mL
1 mM EDTA
0.07306 g
Preparation of Solutions
You should prepare cell buffers according to the following table. All of these solutions should be in stocks that will need to be diluted to get the right concentration. The volume of buffer required varies from around .5 mL to 15 mL depending on the amount of cells and the concentration required.
Cell Buffer
5
250 mM Sucrose
1.28 g
2 mM MgCl2
0.002856 g
20 mM HEPES pH 7.5
1 mM EDTA
* 1 mM PMSF
* 5X PIN (Protease Inhibitor cocktail)
* 1 mM DTT
* These components should be added fresh when you are ready to use the cell buffer.
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.
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.
Harvesting Cells:
You will start with either adherent or non-adherent cells in a petri-dish or a flask with media.
Using a rubber policeman scrape the cells from the plate into their media. If cells are suspension cells this step is not required.
Place the cells with their media into a Falcon tube. Pellet the cells in a clinical centrifuge set on 3 (mcf-7) or 6 (rat-1) for 3 minutes.
Remove the supernatant (discard appropriately). Resuspend the cells in 15 mls of Cell buffer.
Pellet the cells again in the clinical centrifuge at the same speed and duration as above. Repeat this washing step.
Finally resuspend the cells in an volume of Cell buffer depending on the amount of cells.
Nitrogen Cavitation:
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.
*The pressure varies depending on the cell line
Centrifugation:
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.
Clean-up:
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.
