nuclear extract - contamination cytoplasmic/nuclear extrac (Jan/30/2003 )
Hi! I am trying to assay by western blotting NFkB, a trascription factor that translocate to the nucleus, in the brain. I need both cytoplasmic and nuclear extracts.
I have always problems of contamination of the nuclear extract with cytoplasmic fraction. What can I do? and what is the critical step?
In our lab to obtain nuclear extracts and cytoplasmic extracts we use a nuclear extract kit from active motif and has not given us any contamination. Try it
I am also having trouble with my preps of nuclear proteins from thymocytes taken fresh from the mice.
I am using the non-detergent method: swell cells in hypotonic buffer, break cell membrane with dounce homogenizer, slow spin to separate nuclei from cytoplasm, fast spin to get rid of any remaining cytoplasm, resuspend nuclei in high-salt buffer and extract for half an hour, then dialysis.
My main problem has been cells & nuclei lysing prematurely. I am thinking that the amount of hypotonic buffer is critical. I've reduced the amount I add by 80% but still have the problem of cells lysing before they should. I don't understand why swelling is needed anyway . . . wouldn't the mechanical disruption break the cell membrane anyway?
I always get loose DNA in my nuclear pellet, which complicates the resuspension. And I haven't been able to get any protein out of it.
I am keeping everything on ice and using protease inhibitors added just before using buffers. I had this protocol working fine for a few times, then started running into problems. I was not doing anything different, as far as I can tell.
So, any pros out there who can enlighten me? What are the key elements to this thing? How can I stop nuclei from lysing prematurely, and/or how can I save the prep if this happens?
Thanks in advance. And don't tell me to use a kit because that is not an option for me.
Nobody does this without a kit nowadays?!
I have isolate nuc. preps both with a Dounce homogenizer and using .1% NP40.
I find that swelling the cells (10 million or less) for 10 minutes in 400 microlitre of hypotonic buffer, followed by lysis with detergent gives the best result. To make the prep nice and clean, wash the intact nuclei once with PBS (+protease inhibitors) and respin before adding high salt buffer.
This method has been published and works very well.
Some people do a quick sonication to reduce the viscosity of the chromatin. Be VERY careful if doing this. A QUICK sonication should suffice.
In terms of douncing, I dunno, I can never get ALL the cells to lyse without dettergent.
the protocol i use works fine in my hands.It was discussed here. After getting the cytoplasmic fraction, it always remains a few of cytoplasmic extract (cause i don't want to spoil my cytoplasmic extract). Usually, i pippet what remains, and throw it to garbage. But i've never though about washing it in PBS... seems a very good idea.
mikew : may you give e the reference of publication you've mentionned? Thanks in advance
I guess a self-cite is better than no cites.
The protocol is in Blood, july, 2003, pages 237-45 by Witcher et al.
Just add a wash step with PBS after pelleting the nuclei.
I just want to ask a simple question: How can you check the cytoplasmic impurities in the nuclear extract?
I'm now using a non-detergent method in extracting nuclear extract. Eventhough I had increased the number of cells in the sample, the yield was still very low, and the protein concentration of nuclear extract was similar with the cytoplasmic fraction. I doubt that they are all cytoplasmic extract...
A western for GRB2, or B-tubulin should show up only in cytoplasmic extracts.
Nuclear fraction can be detected by lamin B or c-jun.
I'm sure there are other examples.