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Contaminating proteins for Fluorescence polarization assay


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#1 amano

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Posted 15 May 2009 - 12:31 PM

I am handing over some protein samples to a collaborator to do a fluorescence polarization assay to determine binding affinity as a check for activity of a protein that I'm modifying. Using quantitative Western or Coomassie Blue stain, I can determine the concentration of just the protein of interest in a mixture. My question is whether contaminating proteins (carrier protein, enzyme for modifications, etc) will affect the readout of the fluorescence polarization reading. I will lose a significant amount of modified protein if I purify it from the mixture, so I'm wondering if the "dirty" prep would work for fluorescence polarization if I know the concentration of the protein I'm interested in.

Thanks

#2 mdfenko

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Posted 18 May 2009 - 08:29 AM

i really don't know anything about fluorescence polarization but...

i would imagine that it would have similar requirements as methods like x-ray diffraction which requires protein as pure as you can make it (and crystallized, but i don't think you need that).

the best way to find out is to ask the lab which is going to perform the test. they will tell you the requirements.
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