nuclear extraction insoluble pellet problem - here it is again! (Jan/10/2011 )

i know this has been discussed many times in the archive but I just want to double check. I actually posted this in the wrong forum it seems.

Basically i am using Active Motif's nuclear extraction protocol and I am working with 3T3 and DLD-1 cells.

My problem is basically I get an insoluble pellet after I extract the cytoplasmic fraction and attempt to resuspend the pellet in their "complete lysis buffer." I have been able to resuspend it only a few times. I don't understand why most of the time i get this sticky pellet. From reading the archives I understand that this pellet is most likely prematurely ruptured nuclei. So I am thinking two things:

1. Perhaps I am too vigorous with the pipetting of the whole cells in hypotonic buffer that they lyse, but I doubt pipetting could cause this. After incubation with hypotonic buffer for 15 min, I am suppose to add detergent and vortex for 10 seconds. Maybe that could cause the premature lysis? However the next step is to centrifuge at 14,000xg which I believe would certainly cause the lysis. Why would ActiveMotif want this as the cytoplasmic and nuclei separation step? (in their troubleshooting they just say to vortex thoroughly if there is a persistent pellet)

2. Other people have said to just sonicate that pellet. I am concerned about heat causing protein degradation but even with the pellet I still get protein in the lysis buffer. I have not determined how much of it is nuclear protein but I'm sure that sticky pellet reduces my yield by trapping nuclear proteins.


Since i am using a commercial kit I cannot tell what materials I am using unfortunately. Right now I am thinking I should try centrifugation of the cytoplasmic lysate at 1500 - 5000xg instead of 14,000g. I have gotten it to solubilize 3x before so i'm just perplexed what is going on. I do have to mention I am using trypsin and not cell scraping but the first time it worked was with cell scraping so I don't think that's the reason.


Thanks!

still having no luck with this. it is so frustrating especially since I thought buying a commercial kit would simplify things.

i am still getting a sticky pellet. the company still suggests i use dounce homogenizer as the panacea. i think maybe the problem is maybe the trypsin is not completely inactivated or my cell pellet has lysed cells/debris or left over PBS salt that prevents a true hypotonic environment.

will test it tonight. fingers crossed. it's already been over a month of trial and error.

azrael201 on Wed Jan 19 17:32:39 2011 said: