Hypo-osmotic lysisbuffer for "purification" of mitochondria.
Posted 06 September 2012 - 02:41 AM
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
Posted 06 September 2012 - 01:14 PM
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.
Posted 07 September 2012 - 01:25 AM
I know that you get a whole cell lysate in that case, that was exactly why I wanted to remove cytosolic protein first :-)
Posted 24 September 2012 - 04:53 AM
If you have personal experience with it I would like to know.
Posted 24 September 2012 - 12:48 PM
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...
Posted 27 September 2012 - 12:26 AM
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 .-)