nuclear extraction question - (Dec/10/2011 )
Recently I did a nuclear extraction of my cells with the Cayman Chemical protocol here: http://www.caymanchem.com/pdfs/10009277.pdf
It was getting very late so after resuspending the pellet in the Complete nuclear extraction buffer (the pellet was still intact) i froze the samples at -80 C.
Are these still salvageable - i.e. if I thawed them would I be able to just continue the steps as detailed in the protocol - or should I throw these away? Any tips on this would be much appreciated.
Thanks in advance!
You froze at step 8? If so, your cells will be lysed and it is unlikely you will get a clean nuclear prep, if any.
I wonder of why a clean nuclear prep won't be obtained when the nuclear pellet is being frozen after the cytoplasmic fraction was removed? I can see that no separation will be happening if one freeze the cell pellet without prior removal of the cytoplasmic extract. But if the cytoplasmic extract are already removed, then the nuclear extract will likely be already separated at this stage, and just need to be lysed by the hypertonic buffer, thus it will be fine if they are frozen at this stage?
As far as I can tell from the protocol, at this point the nuclear preparation is not clean, there are still the nuclear membranes and associated proteins left behind, as well as probably a lot of the cytoplasm - the previous step was just a quick spin to remove debris. However, on closer examination of the protocol, they might be OK, I don't know why the OP didn't spend the extra 10-15 min to finish the protocol, which would have made this point moot.
Hi Bob1, the reason I ask is because I have often done this since the next nuclear lysis protocol for me involves 40 more minutes of lysis with vortex every 10 minutes. My application is to separate transcription factors that may shuttle between cytoplasmic and nuclear extract. I also usually add another PBS rinse to my nuclear extract to ensure that I remove my cytosolic fraction before freezing. I hope this is ok?
That should be OK, but I would definitely do the rinse before freezing. Personally I am not a fan of these sorts of nuclear extractions, as I don't find that they are clean enough for my purposes - my protein shows a very peri-nuclear distribution, so my nuclear preps have to be very clean before I can definitively say that there is nuclear shuttling or not.