nuclear extraction question
Posted 10 December 2011 - 12:44 PM
Recently I did a nuclear extraction of my cells with the Cayman Chemical protocol here: http://www.caymanche...fs/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!
Posted 11 December 2011 - 12:21 PM
Posted 11 December 2011 - 04:34 PM
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?
Posted 12 December 2011 - 12:07 PM
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.
Posted 12 December 2011 - 12:15 PM
Posted 13 December 2011 - 11:41 AM