Contaminating proteins for Fluorescence polarization assay - (May/15/2009 )
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.
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.