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Protein degradation SDS PAGE

protein degradation SDS

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5 replies to this topic

#1 biochem06

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Posted 08 July 2014 - 07:34 AM

Hi,

 

Just wondering doe sthis look like the protein has degraded? standard 12% SDS PAGE gel run under standard reducing conditions.

 

The samples are blood and plasma.

 

As you can see there are no nice defined bands, just smearing and then those big bands at the bottom which I suspect could be all the degraded protein?

 

Any suggestions would be great!

 

 

Attached Thumbnails

  • gel.jpg


#2 bob1

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Posted 08 July 2014 - 12:47 PM

Could be a number of things besides protein degradation - incorrect pH of the gel or the loading buffer, incomplete denaturation, old SDS, not buffered properly, gel run too hot...



#3 biochem06

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Posted 09 July 2014 - 12:20 AM

Hi bob1 thanks for your reply. I've been doing these gels for years and this is the first time I've had gels like this - albeit it's the first time I've used blood/plasma. The gel was made up freshly with fresh buffer etc, heated for 10 mins at 95C in gel reducing buffer, the SDS was newly made up and the temp of the gel was fine throughout - this is why I was wondering if the sample has degraded? It's been in the freezer 6 months and I would say has been freeze-thawed at least twice

 

thanks



#4 mdfenko

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Posted 09 July 2014 - 06:01 AM

can you compare these samples with fresh blood/plasma?

 

-20 or -80 freezer?

 

proteolysis can occur at -20C. what inhibitors have been added prior to freezing?


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#5 mlomonaco

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Posted 09 July 2014 - 07:41 AM

The bands are vWF multimers.  They are a proxy for your protein NOT being  degraded!



#6 biochem06

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Posted 21 July 2014 - 12:37 AM

Hi mlomonaco, that it interesting and something I have never come across before. Do you know what I need to do to my samples in order to get the gels to look more like "typical" SDS PAGE gels i.e. many discretely resolved bands instead of these large bands?






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