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Staining SDS-PAGE gel with EtBr?


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#1 Fzang

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Posted 30 December 2014 - 10:37 AM

I want to show the presence of small DNA strands attached to proteins of various sizes, as these proteins seem to stain very poorly in coomassie compared with no-DNA proteins.

Can I simply run a normal SDS-PAGE and afterwards stain it in EtBr (in ethanol and acetic acid?) for a while and UV it? If so, what would be an adequate amount of EtBr to add? I haven't worked with DNA for quite some time.



#2 bob1

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Posted 31 December 2014 - 12:54 AM

No, SDS-PAGE will separate proteins but not DNA effectively. You should run the gel with TBE or TAE or SB (or LB) in the place of the conventional tris-glycine buffer system and leave out the SDS.



#3 merlav

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Posted 31 December 2014 - 10:54 AM

Just prepare the acrylamide-bis in TBE 0.5-1X (I prefer TBE for small strands like oligos) add APS and TEMED.  Run the gel in TBE (same concentration that you use for the gel.  You can add a microliter of EtBr to the sample so you don't have to stain it after running were it could tear.  If possible add to one of the plates a repel solution. 


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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
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#4 Fzang

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Posted 31 December 2014 - 11:58 PM

The proteins are linked covalently to a small DNA strand about 1/20th of the MW of the protein. If I changed the buffers wouldn't that mess with how the proteins run on the gel? The gel will not be containing any free DNA.

The problem appears to be that the DNA barely has any influence in how the protein runs, and sometimes seems to "repel" coomassie when staining.

I have measured concentrations using either UV-vis or BCA assay and afterwards detected enzymatic activity of proteins at equal concentrations. In both cases I get the same result; slower kinetics for the DNA-coupled protein. However, this is only true if my measured concentrations are correct.

Then why is it almost impossible for me to reproduce my SDS-PAGE results reliably? Sometimes the protein-DNA runs differently, sometimes it doesn't. Sometimes it stains, sometimes it doesn't. I'm heating my samples for exactly 5 minutes at 95 °C every time, before running.

I don't know if I'm doing something systematically wrong every time.



#5 phage434

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Posted 01 January 2015 - 08:36 AM

Can you label the DNA fragment with a covalently attached fluorescent probe? You wouldn't need to dye the gel at all if you can.



#6 merlav

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Posted 02 January 2015 - 01:48 PM

Oh Sorry I misunderstood. No you can't change the buffers it most remain as a regular SDS. The EtBr attach to the DNA grooves. If the protein is bond the DNA, the grooves still free for the EtBr? Don't know but I do like phage approach.
Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#7 Fzang

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Posted 02 January 2015 - 06:51 PM

Thanks for all the suggestions. I'll go to the lab these days and try out some things. Then I'll let you know if I've had any success. tongue.png


Edited by Fzang, 02 January 2015 - 06:56 PM.





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