Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

what nuclei look like after high salt nuclear extraction - (May/30/2007 )

Pages: 1 2 Next

Thanks for your viewing.

I am trying to extract nuclear proteins from myotubes for the purpose of protein complex purification. I did regular hypotonic buffer followed by high salt extraction of nuclear protein. After I added high salt buffer (HEPES, with 420mM KCl) and rock in 4C for 1h, I expected to see nuclei are destroyed. But when I inspected it (stained with Trypan blue), I found large number of cell nuclei are still there. It seems that they are intact. (they were stained with trypan blue). Is this normal? Should I see nuclei totally destroyed without normal shape of nuclei? Is it possible that the nuclear protein has been extracted and the likely-intact nuclei that I saw is only the membrane part of the nuclei or some insoluble proteins?

Thanks again.


the disruption of nuclei occurs by the addition of high salted buffer AND the centrifugation at 20000g for 45'....


Thanks for the answer. Really appreciate it.
I have tried to centrifuge it (15500g, maybe the speed is not high enough?), I got a huge pellet in 1.5ml eppendorff tube. Then I resuspend the pellet with buffer containing 150mM NaCl and 0.5%NP40, and rock for 30 min. I was trying to see if detergent could be helpful to disrupt the nuclei. Oops, the nuclei are still there (under trypen blue staining)!!! then I spin down the pellet again. It seems that the pellet is smaller this time. Any further inputs?

QUOTE (fred_33 @ May 31 2007, 03:13 AM)
the disruption of nuclei occurs by the addition of high salted buffer AND the centrifugation at 20000g for 45'....


what are the volumes of nuceli and for buffer used ?
i mean cells should be lysed in 2volumes of the cell pellet and after nuclei are disrupted by 1.5 vol of high salt buffer aren't they ?


I used 4x volumes of initial cell pellet for hypotonic and 2Xvolumes of nuclei pellet of high salt.

I should update my last post a little. As I wrote, after I lyse the nuclei in 0.5% NP40 lysis buffer, I can still see nulcei. But after I spin down the lysate in 15000g, I only got a small pellet, and it is hard to resuspend it any more (it is sticky). When I took some to inspect, I cannot see ANY nuclei. So I think the spinning down the lysate can really help to break down the nuclei, though I still don't know why the high salt buffer was not working well. (it may partially work as I can get around 700-800ug of protein from the high salt fraction--15cm Plate). After I lyse the nuclei using NP40 buffer, I got another about 1mg of protein in the buffer.

What I am trying to figure out is how to use high salt to efficiently lyse the nuclei. I want to do mass-spec, and it preferred to not to have detergent.

Thanks a lot.

QUOTE (fred_33 @ Jun 1 2007, 07:16 AM)
what are the volumes of nuceli and for buffer used ?
i mean cells should be lysed in 2volumes of the cell pellet and after nuclei are disrupted by 1.5 vol of high salt buffer aren't they ?


well my protocol is 0.1%NP40 (so i guess you meant 0.05%NP40?)
i don't see a bi difference in my hands between 0.05 and 0.1% NP40 (except for in vitro transcription assays where 0.1% is better)
so if you see the nuclei after it's ok.
After the lysis of cytoplasm 5000rpm during 15' allow the nuclei to pellet. More speed breaks nuclei !
2things come to my mind :
after spinning down 15000g, the viscous thing is composed only of membranes, DNA and histones that stuck very well to DNA. (i've discussed of that with my bos to check)
if you spin down by 5000g you'll get a pellet needed to be resupended by pipetting up and down.

my protocol uses glycerol for breaking the nuclei

I use protocol described here
Now i do a second wash with hypotonic A buffer and don't include NP40.
It helps remove last cytoplasmic proteins and get out NP40.

You may try it for your exp ? unsure.gif


Thanks for the input and the protocol.

Sorry, I did use 0.5% NP40, not 0.05%, and I used it in the buffer for lysing the nuclei, not the first step hypotonic buffer. Will this (high NP40 concentration) cause any trouble? I have limited experience, so appreciate if you can let me know.

What I am thinking right now: I can use hypotonic buffer to get nuclei exposed (I was fine until this step), and then use NP40 buffer (containing 170mM NaCl, 0.5% NP40, EDTA, Tris) to lyse the nuclei. Do U think this will lyse nuclei better, and give me more protein extracted? I want to do mass-spec after purification. Detergent might not be good for mass-spec, but I will try to wash it away from my immunoprecipitated samples. Does this make any sense?

BTW, what is the principle behind "glycerol breaking down nuclei"? How glycerol does it?

Thank you again for the help.


not the first step hypotonic buffer
do you mean you rouhly do only buffers B and C step? i mean how do you get rid of cytoplasmic protéins?
Will this (high NP40 concentration) cause any trouble?
i don't think so. The NP40 is used in limited concentration in my protocol in order to lyse cell membrane only, and not the nucleus membrane. so 0.5% would soubilise all membranes i think.
NP40 buffer (containing 170mM NaCl, 0.5% NP40, EDTA, Tris)
normally would break up nuclei
glycerol breaking down nuclei
i don't know... where did you see that ? i wiant to read the article to understand the point behind this (if it's different of the glycerol-based solutions, added only for better cryopreservation at -80° of the proteins


I still used buffer A (hypotonic buffer), but did not add the NP40 into the hypotonic buffer.

For the glycerol thing, I saw a sentence in your last post: "my protocol uses glycerol for breaking the nuclei". Because you mentioned this, so I was wondering how you can break the nuclei by glycerol?

I am glad to chart with you! THANKS A LOT. I am going to try the NP40 lysis and let you know how it works.


i replyed :
"when doing a great volume of nuclear extraction, i add the buffer, resuspend very well by pipetting up and down, and then when it's well done, i vortex the nuclei pellet, tube opened, and i add drop by drop the NP40. I let vortex roughly 10'' after finishing addition of np40.
Then go for your rocking of the "resuspension" but go til 45'. 20 000g is ok, but at least for 30'.

if that goes bad, i would suggest you to try my protocol. I would say nuclear proteins are done without NP40. I did affinity purifications very well by that extraction method.

then georges sent :
thank you for your suggestion. I really appreciate it. I have read your protocol and there are few follow up questions. I am really grateful if you could help again.

1. what does the first paragraph mean (Sometimes, i get part of pellet in the eppy...)? Is it important? Eppy stands for eppendorf tube, right?

2. in the next few steps, you pellet the cell twice (first by 1000rpm, thentransfer the cell pellet into eppys and pellet again by 2000rpm). Is it important to do everything in eppys? As I have large vol, of cell, can I use 15ml conical tube? What is the rcf of 1000rpm for the rotor that you used?

3. Buffer A: PIC stands for protease inhibitor cocktail, right?

4. What is the stock concentration of NP40 buffer that you used? Should I also add it drop by drop to the buffer A after you resuspend the cell pellet? I think drop by drop while vortexing could be very helpful to break down cells.

Thanks again for your help.



Pages: 1 2 Next