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protein staining with EtBr!!

EMSA gel shift ethidium bromide protein protein staining

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4 replies to this topic

#1 Shrubal

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Posted 06 September 2015 - 06:34 AM

I'm a newbie at EMSA and am trying to get this to work for me. So I carry out my reaction, load &run the gel and the stain it with ethidium bromide in order to stain the DNA. However, as in the attached picture, I'm getting smears and this is seen in the no DNA control as well. Can anyone help answer why a lane which contains only protein is being stained by EtBr? I've run the protein on an SDS-PAGE followed by colloidal coomassie staining and I see no degradation of the protein.

Thanks!

 

Attached Thumbnails

  • EMSA_21aug2015.png


#2 mdfenko

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Posted 08 September 2015 - 03:42 AM

presumably, the protein binds dna. you could be seeing dna carried by the protein or dna from dust or other contaminant.


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#3 dpo

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Posted 08 September 2015 - 04:15 AM

Where does the protein come from? If this is produced in bacteria, you may have co-purified bacterial genomic DNA together with your protein.



#4 neuron

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Posted 09 September 2015 - 02:04 AM

I don't have any experience with EMSA but I was also facing same problem few days back, when I was trying to see RNA in denatured protein complex. My protein complex used to give me band on agarose gel. Then I searched and found out that even protein get stained with EtBr. Here is the link-

 

http://ac.els-cdn.co...fccd4e627d24ae8



#5 mdfenko

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Posted 09 September 2015 - 03:08 AM

nice paper, neuron. the only problem is that the paper says staining requires sds, however, the gel used in emsa does not use sds.


talent does what it can
genius does what it must
i do what i get paid to do





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