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Regarding SDS PAGE of an oligomeric protein


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#1 sify

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Posted 20 June 2011 - 10:01 AM

Hello friends!
I am working with an oligomeric protein. I am using 9M urea to denature it but surprisingly when I do SDS PAGE of denatured protein i find a band of dimer and a monomer in the proportion 1:3 in intensity.
But when I do NMR of the denatured protein I get clear spectrum where all the amino acids are visible , which suggests that the protein is completely denatured ,i,e. there is no dimer present. This is very puzzling fro me.
What do you think can be reason for this?
Please give your suggestion on this.

Edited by sify, 20 June 2011 - 10:02 AM.


#2 protolder

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Posted 20 June 2011 - 09:40 PM

Hello friends!
I am working with an oligomeric protein. I am using 9M urea to denature it but surprisingly when I do SDS PAGE of denatured protein i find a band of dimer and a monomer in the proportion 1:3 in intensity.
But when I do NMR of the denatured protein I get clear spectrum where all the amino acids are visible , which suggests that the protein is completely denatured ,i,e. there is no dimer present. This is very puzzling fro me.
What do you think can be reason for this?
Please give your suggestion on this.

Hola, the sample treatment for SDS-PAGE you know is boiling with reducer and detergent, probably if you made somethig similar you will have the same species , moreover if the multimerization is S-S mediated. Think in broke the complexes with reducer +/- urea and made non reducer SDS-PAGE where samples are treated only with SDS withouth boiling. This is a way to see multimers as the case of some membrane receptors. Buena suerte

#3 chabraha

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Posted 24 June 2011 - 09:28 AM

Just a thought..........but could the Urea be getting diluted in the gel/buffer allowing an "in gel" interaction?
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#4 mdfenko

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Posted 14 July 2011 - 12:12 PM

Just a thought..........but could the Urea be getting diluted in the gel/buffer allowing an "in gel" interaction?


there won't be much urea left after boiling (although the protein may become carbamylated). but the protein will be denatured by the sds and reducing agent.
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