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Mystery of SDS gel, working with the protein GB1 (T2Q mutation)

GB1 SDS gel protein

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#1 Tom Aharoni

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Posted 31 March 2015 - 12:47 AM

Hey!
 
I used SDS gel in order to validate the presence of the GB1 protein (6.22kDA) in my sample before and after heating (in order to lysis of the cells) & cleaning with gel filtration column.
 
It is known that the GB1 domain runs at roughly 13.5kDa despite being only 6.2KDa, so the location is logic.
 
The mystery is that after the cleaning, there are impurities of allegedly two proteins, a little bit higher and a little bit lower the original concentrated GB1, that don't appear before cleaning.
 
Could anyone please explain how is it possible? 
 
Thanks,
 
Tom.

Attached Thumbnails

  • 1.2.2015 - minimal media with IPTG low.jpg
  • after cleaning low.jpg


#2 mdfenko

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Posted 31 March 2015 - 04:20 AM

a couple of possibilities:

 

aggregation and decomposition

 

concentration of contaminants


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#3 Tom Aharoni

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Posted 31 March 2015 - 04:48 AM

Thank you!

 

Is there a different kind of gel that could prevent the protein from aggregation and decomposition? native gel maybe?



#4 mdfenko

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Posted 31 March 2015 - 05:13 AM

decomposition would occur during handling.

 

aggregation can occur during handling and/or during heat treatment with sds sample buffer. if it is occurring during the heat treatment then you can incubate at 60-70C instead of 95-100C.

 

native page will not reduce either and may actually increase aggregation.


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




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