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Bradford assay


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#1 Mariam

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Posted 02 September 2013 - 09:10 AM

Hi,

Since I have purified my protein of the interest using denaturing method, I had to get rid of urea before mice injection. I tried to dialyze my protein against reducing concentrations of urea in PBS. The dialysis has been done successfully as no aggregation happened. But when checking the protein concentration using Bradford assay, not only color change was not happened by adding the protein sample to Bradford reagent but also the concentration reported by the spectrophotometer was zero! However, after running the dialyzed protein on the SDS-PAGE the band of protein was visible! There was no detergent or other Bradford interferers in the protein elution buffer or in PBS. Could anyone help me with this problem?

Thanks 

 

Edited to make the font smaller - bob.



#2 mdfenko

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Posted 02 September 2013 - 03:10 PM

what is the size of the band? how intense is it?

 

you can try other methods to determine protein concentration (bca, lowry, etc).

 

it's possible that the concentration is too low for accurate determination, the blank could have been contaminated, etc.


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#3 Mariam

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Posted 07 September 2013 - 09:14 AM

what is the size of the band? how intense is it?

 

you can try other methods to determine protein concentration (bca, lowry, etc).

 

it's possible that the concentration is too low for accurate determination, the blank could have been contaminated, etc.

Thanks for your tips. The size of the band is 25kD as expected and it is intensive. I have done the bradford assay before dialysis and the result was ok sad.png






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