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several bands in non-reducing SDS-page gel - protein without cysteines

non-reducing gel

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#1 sereb

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Posted 13 October 2014 - 02:12 PM

Hi everybody,

 

I have a protein which does NOT have any cysteine. I know that it does form oligomeric structures and I hypothesised the complexes formed through non covalent bonds.

 

If I boil the sampIe and add a reducing agent into the loading buffer I get just one band (monomeric form).

 

If I run a non-reducing SDS-page gel using a sample loading buffer without beta-mercaptoethanol, I get several bands (corresponding to dimers and trimers sizes).

 

I knew that the difference between the two type of gels was that in the non-reducing gel the disulfide bonds are not destroyed so, if there are oligomeric structures, you can detect them.

But I don't have any cysteine and I thought boiling can destroy non-covalent bonds.

 

So, I did expect not to see any difference.

 

The question is: why I did get several bands in my non reducing gel even after boiling the samples at 95 C for 5 min?

 

Does anyone has any experience in this field and is willing to help?

 

I would really appreciate cause it's the first time I'm trying to address a similar question and I'm kind of lost.

 

Thank you in advance

 

 

 

 

 

 



#2 mdfenko

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Posted 14 October 2014 - 03:51 AM

you could be seeing aggregation caused by incubation at 95C. you can check for this by:

 

1) reducing the incubation time

2) reducing the incubation temperature (60-70C for 10-20 minutes)

3) omitting the incubation (you don't need the added energy to break sulfhydryl bonds)


Edited by mdfenko, 14 October 2014 - 03:52 AM.

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

#3 sereb

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Posted 14 October 2014 - 07:59 AM

Thank you!

This is very helpful :-)






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