With this type of behavior, one suspicion is that you didn’t let the reaction 
come to equilibrium prior to taking the measurement and the variability between 
time of mixing and measuring between individual replicates is introducing extra 
variability. Take a concentration at which you know there is a significant 
signal change and measure the polarization every few minutes for an hour. You 
might find that you need to pre-incubate the samples for an extended time prior 
to taking measurements.

With that said, my first suspicion is that you have lousy binding.

Eric


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Friday, July 21, 2017 9:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all,

I am trying to measure the difference in polarization upon the binding of the 
DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions 
of my protein to it (100 microM to 1 nM). I do get a decrease in difference of 
polarization with decrease in protein concentration. However, the results are 
difficult to reproduce and also vary greatly within triplicates of an 
experiment.

Similar observations have been observed by my colleagues with their proteins.

Are there any tips or precautions to keep in mind while setting up these 
reactions?

Looking forward for suggestions.

Thank you.

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