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Nuclear Extraction headache - persistent pellet glob


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#1 azrael201

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Posted 30 December 2010 - 09:58 AM

I have been trying to use Active Motif's nuclear extraction protocol and kit but i keep running into the same problem with three different cells lines. I had a previous thread about my cell scraping woes which I think I have overcame but now I have a new problem interesting problem...

After I scrape and collect my cells I spin down and discard the supernatant and then I suspend that pellet in hypotonic buffer for 15 min to lyse the cell membrane. I then add detergent (as per protocol) and then vortex and spin down at 11,000x g for 30 seconds (protocol says 14,000x g for 30 sec). I save the supernatant as my cytoplasmic fraction.

Next is to collect the nuclear fraction by resuspending that pellet in a complete lysis buffer. This is my problem: I cannot resuspend that pellet! no matter how hard i vortex or pipette. The pellet is sticky and it actually jams the pipette tip. Troubleshooting in ActiveMotif manual just says to vortex thoroughly-needless to say that didn't work at all.

My postdoc believes this could just be DNA sticking together. Should I be worried that I would have this sticky pellet? I feel like since I can't resuspend that pellet I am compromising my complete lysis of nuclear membranes and reducing the yield.

Could this all be from DNA broken during the cell scraping? I'm going to try trypsinizing today.

#2 scolix

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Posted 30 December 2010 - 10:06 AM

I would use sds buffer to make sure you have the complete lysis of nuclear membranes. Any time you lyse nucleus, its going to be sticky because of DNA.

#3 azrael201

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Posted 30 December 2010 - 12:13 PM

I would use sds buffer to make sure you have the complete lysis of nuclear membranes. Any time you lyse nucleus, its going to be sticky because of DNA.



the problem is it was probably sticky before i added the complete lysis buffer. i can't really tell for sure but basically after i remove the cytoplasmic fraction i add complete lysis buffer to the pellet and try to resuspend by pipetting up and down. however, i can't because it is too sticky.

#4 bob1

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Posted 04 January 2011 - 12:54 PM

The stickiness indicates that the lysis is working. You could try sonicating to break the DNA.

#5 azrael201

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Posted 07 January 2011 - 04:39 PM

i was considering sonicating it. hmmmm maybe i will go for it.

it's just strange because it seems to work with my fibroblast cell line before but today it clumped up for the fibroblast cell line. I counted the cells and made sure I had the right proportion of reagents too. I did allow the cells to sit in hypotonic solution extra long to break the cell membrane but i don't think that should have made a difference.

I added extra lysis buffer to break the clump and also extra DTT in case that is all clumped protein. It did not work.

I'm sure sonicating will break it up but i'm just perplexed why sometimes I get this clump after spinning down and sometimes I don't. Could it be I didn't spin down long enough? (instructions ask for 14,000xg but my centrifuge only hits around 13,000xg so i let it spin down for only a couple more seconds) it would seem counter intuitive.

also would sonicating affect my yield?

Edited by azrael201, 07 January 2011 - 04:39 PM.


#6 zienpiggie

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Posted 18 January 2011 - 09:04 PM

Did you try measuring the protein content anyway?

#7 azrael201

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Posted 18 January 2011 - 09:22 PM

i did. still need to figure out how to measure it with the commercial lysis detergent.

it's still giving me a major headache even though Active Motif is sending me all their support documents...

apparently a dounce homogenizer is the end all be all...it has worked 4 out of ten without douncing and i'm still a month into this not sure when it works and when it doesn't. The company says it's because i don't have cell lysis prior to centrifugation and recommended the dounce. I just don't believe there is not a more elegant solution with just hypotonic solution and detergents.

how can i optimize the hypotonic buffer and detergents?

#8 jonas albarnaz

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Posted 13 March 2011 - 09:47 PM

Azrael201,

I have the same problem. I wrote a topic here about it. When you solve your problem, please let me know.
Also, I'd suggest you do not vortex sample after adding detergent, to avoid nucleus lysis and contamination of your cytoplasmic fraction.

Best,
Jonas

#9 bob1

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Posted 14 March 2011 - 12:29 PM

The type of dounce is very important - you want one with a ball shaped head on the pestle and a clearance of 50 nm or so (IIRC), not the tubular shaped one with the ground glass sides.

#10 azrael201

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Posted 14 March 2011 - 01:52 PM

the dounce i am ordering from VWR is on backordered til the 27th.

i will let you guys know then.




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