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Nucleic Acid interference in Western Blot / Optimal Nuclear Extract Protocol


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4 replies to this topic

#1 scs

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Posted 09 November 2010 - 08:37 AM

I wanted to look into a new Nuclear Extract protocol for mammalian cell lines used to generate lysates for Western Blot.
We test our antibodies via Western Blot
We have some cell lines that yield very low concentrations of nuclear proteins such as A431
I came across some protocols that remove nucleic acids from the nuclear protein extract.

1) Is there any interference on SDS-page with the presence of nuclear acids in the nuclear extract?
2) Is there any reasons why I should remove nucleic acid from our nuclear extracts?
3) Is there any ideal protocol / set of reagents that really can maximize the amount of nuclear proteins that can be extracted from mammalian cell lines?
(A protocol where someone has really noticed significant gain compared to other protocols)
4) We dialyze our product at the end of the protocol, but I have read that this is not optimal. Is this true?

Thank you so much for the help.

Edited by scs, 09 November 2010 - 08:41 AM.


#2 ksturm

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Posted 09 November 2010 - 10:40 AM

Sometimes DNA and RNA from cell extracts form slime, which is hard to load on gel and make problems while running. If this is your problem, treat samples with DNAseI and RNAse A. Both enzymes work even in 0,3% SDS. Please find more detailed instructions in Millipore UserGuide for 2-D-electrophoresis.
What dialysis do you use?

#3 scs

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Posted 09 November 2010 - 01:30 PM

Thanks so much for the response.

We dialyze in solution containing:

DTT
PMSF
Hepes
Glycerol
KCl
EDTA

and we use:

Thermo Slide-A-Lyzer Dialysis Cassettes

10,000 MWCO

We are not having the "slime" issue.

I just saw a protocol that has additional steps to remove nucleic acids. I believe that the additional steps may reduce overall nuclear protein recovery compared to another protocol (fewer steps) that do not include nucleic acid removal.

Is this correct thinking?

I would run a side by side but wish to not focus on this and move forward with picking most optimal protocol that recovers the greatest amount of nuclear proteins.

Thank you again for helping and let me know if you have any further ideas.

#4 ksturm

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Posted 09 November 2010 - 11:47 PM

Dialysis can cause protein loss, because of unspecific binding of protein to membrane. If you don't want to leave dialysis, BSA added to sample solution prior dialysis could prevent this loss. Actually I cant provide a comparison between particular protocols. But I had good experience with nuclear extract kits (f.e. Active Motif). This is designed to prepare nuclear proteins for Western. protein yield in my experiments were always sufficient.

#5 scs

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Posted 10 November 2010 - 08:19 AM

Thank you again for your help on this. We just tried adding a sonication step before the final spin and our yield jumped up 3X.
Amazing. I will also try the BSA prior to dialysis as well and see what yield can be obtained. Thank you again so very much.




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