Does anyone know if it is possible to achieve sub cellular (nuclear, mitochondria, cytoplasm) enriched fractions from frozen cell pellets? If so please help me with the protocol and appropriate buffers. I need my proteins for Western Blotting and a protocol without ultracentrifugation.
Do you have a dry pellet or are the cells suspended in any buffer? In the latter case it is sometimes diffcult to get good fractionation because of the effects of the buffer ingredients during freezing.
Without any further information I would suggest to use a hypotonic buffer, e.g. 20 mM Tris-Cl pH 7.4, 50 mM KCl, 5 mM EDTA + protease inhibitors or similar. Use around 10 packed cell volums and a dounce homogenizer for a good suspension. Centrifuge at 500-800x g and repeat 2 times. The final pellet will contain the nuclei. Use the supernatant for centrifugations with increasing g-force (2.000; 10.000; 40.000; 100.000). Without using an ultracentrifuge it is hard to sediment membranes, though.
I got dry pellets - just centrifuged and nitrogen frozen cells
Thanks! Maybe you could suggest some way to achieve mitochondria enriched fraction in such a situation - it is more important to me than nucleus. Which g - force pellet will be the mitochondria enriched one when using the buffer you have suggested.
Hey Tomasz.
I would use a TES-buffer (10mM Tris pH7.4, 1-5mM EDTA, 250mM Sucrose). After Lysis and centrifuging the nuclei as Lars suggested, the Mitochindria should be in the 10.000g Pellet (after 20min). If you want more pure and clean mitochondria, you need to do ultracentrifugation and should resuspend this pellet in TES and put it on top of a sucrose gradient (2-step with 1,5M and 1M). After 60 min at 80.000g the Mitos should be at the interaface of the two sucrose concentrations.
Good luck!
Hi this is how I separate mitochondria proteins without ultracentrifugation. I use a lysis buffer containing 250mM sucrose, 70mM KCl and 2mg/ml digitonin in PBS and then you leave your samples on ice for 5 min and centrifuge at 250g/5min/4°C. The supernatant is removed and stored at – 70°C as the cytosolic fraction. The pellet is ressuspended in immunoprecipitation buffer (100mM Tris-HCl, pH 7.0; 10mM EDTA; 100mM NaF; 10mM sodium pyrophosphate; 10mM sodium orthovanadate and 0.1mM PMSF) and placed on ice for 10min. The suspension is centrifuged at 12.000rpm/10min/4°C and the supernatant is stored as mitochondrial fraction at –70°C. Hope it helps, cheers!
Thank you Jakob and Hilton - have you tested this protocols with frozen cell pellets or only without frozing? And a question about digitonin - could it be substituted by other detergent - or this is the substance that do the trick?We do not have it in lab and it is quite expensive...
Hi Tomasz, you can also use frozen pellets. In fact, when I don't have much time i usually wash my plates with PBS and then freeze it at -70. I never tried but you can use other detergents like saponin or CHAPS.
Thanks Hilton - after re suspension in IP buffer and centrifugation I got the mitochondria enriched fraction - "membrane fraction" in the supernatant but the nuclear enveloped is also destroyed ? I can only separate cytoplasm with this protocol? Ifo So is it possible to insert an additional centrifuging step after addition of the first lysis buffer to collect the nucleus? The nuclear envelope is intact after treatment with first lysis buffer? And my last question is what stays in the pellet after 12000g centrifugation in the last step? Sorry for bothering but I really want to know what I am fractionating and appreciate your help.
I never tried, maybe it is possible... not sure. For the nuclear protein I do a nuclear extraction... but you can try to extract using this buffer: 5mM HEPES, 1.5mM MgCl2, 0.2mM EDTA, 26% glycerol, 300mM NaCl and protease inhibitors, and to make sure you have nuclear protein check the presence/absence of housekeeping such as TBP.
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