Hypo-osmotic lysisbuffer for "purification" of mitochondria. - (Sep/06/2012 )

Hello Everbody.

I would like some help with formulating a new protocol for a rough fractioning of the organel content of some cell culture samples (fibroblasts).

What I currently am doing:
Basically freeze/thaw cycles and a brief waterbath sonification of the whole cell.

What I would like to do:

Harvest cells and lyse them with a hypo osmotic lysis buffer (possibly with some nonionic detergent (and protease inhibitors)). It's essential that the mitochondria should be largely unaffected by this.
Alternatively I have read that 30 min of aggitation at 4 deg celcius in a isotonic lysis buffer can is sufficient to disrupt the plasma membrane, any experience with this?

Then spin them for 5-10 mins at 4 deg celcius. The supernatant should then ideally consist of cytosolic proteins.

Then resuspending the pellet in a mitochondria lysis buffer and using a disruption technique that is rough enough to open both the inner and outer membrane. Here I'm properly back to freeze/thawing, waterbath sonification or a blunt-ended needle and a syringe (the latter option can be done without detergent!?).

A further centrifugation would leave me with a supernatant full of mitochondrial proteins, though the disruption method will properly affect the degree to which there is any of membrane bound proteins left.

Please share your experience with: plasmamembrane disprution (method and buffer) & mitochondrial lysisbuffer.

Sorry for the long winded post, but felt context to be important :-)

Thanks in advance for any advice, sincerely Martin

Freeze/thaw will disrupt all the membranes in the cell - essentially you are just making a whole cell lysate.

The typical protocols are to lyse in a hypo-osmotic buffer, that will swell the cells and pop the plasma membrane. Here is a link to a basic protocol: subcellular fractionation (Abcam). Note step 7.

Thanks for the protocol, will look into it at once.
I know that you get a whole cell lysate in that case, that was exactly why I wanted to remove cytosolic protein first :-)

bob1, I don't know if you keep up with old topics, but if you do please add a comment to that effect as having compared the protocol with others and using a bit of logic I think the protocol you send is a little off the right track.
If you have personal experience with it I would like to know.

Cheers :-)

I havn't used that protocol at all, but it is more or less similar to ones I have used at other work places.

If you want really detailed protocols try "Current Protocols" or "Nature Protocols". The best ones are the ones using gradient centrifugation to purify the components.

As you can see, I do keep up with posts - I use the "view new content" link...

It's just that is seems to skip a lot of clarifying information.
Example:

Step 5. Which supernatants from step 4 is he refering to? I Guess both of the "wash" steps.

Step 6. Centrifuge again, but for how long, both 5 and 10 minutes centrifugation has been used in previous steps.

Step 7 The author wants me to "resuspend in the same buffer as above", only we used two different buffers above (I guess he means the one with glycerol).

I trying it out as soon as my patient fibroblast samples have grown to a suffient total mass. Hope it will work .-)