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Bradford positive but no Protein on SDS PAGE

Bradford SDS-PAGE

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4 replies to this topic

#1 VictoriaTrinkaus

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Posted 21 June 2015 - 08:58 AM

Dear All,

 

I have got a question: At the moment I am trying to express a protein in Rosetta gami 2 (DE3) PLysS. After Induction with IPTG and purification with a Nickel column, I did a Bradford assay, which was positive, both in my wash- and elution-fractions. After that I did a SDS-PAGE with about 20 µg Protein per gel pocket, but found no protein on it after coomassie staining. 

 

Does anyone have a good explanation for this?

 

Thanks


Edited by VictoriaTrinkaus, 21 June 2015 - 09:46 AM.


#2 Micro

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Posted 21 June 2015 - 01:53 PM

In what type of buffer did you elute your proteins from nickel column? Bradford assay results can be effected by some chemicals that are commonly used in the elution buffers. To give accurate results the chemicals may need to be decreased or removed (via de-salting column or dialysis for example) prior to running the assay.



#3 phage434

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Posted 21 June 2015 - 07:02 PM

Did you run a marker lane? Did it stain?



#4 mdfenko

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Posted 22 June 2015 - 05:36 AM

did you use g-250 or r-250 coomassie?

 

g is more specific and won't stain as much as r.

 

you can also try silver staining to detect lower amounts of protein.

 

but the problem is probably what micro said, buffer components that interfere with bradford.


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#5 Kyle Strickland

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Posted 06 September 2015 - 10:12 AM

Also you will find that the Bradford has a lot of problems with most detergents. In my lab we kept having false positives due to Triton in our buffer. I would check each component of your buffer solutions for compatibility of the Bradford assay!






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