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.
