off topic: fluorescence polarization positive control

From: Huiming Li
Date: 7 March 2012 18:21

Hi all,

  Sorry for the off topic question.  Could someone suggest a positive control for FP assay? I am developing this assay and would like to use it for trouble shooting an instrument.

Thanks,

Huiming Li

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From: KAMESH Narasimhan (GIS)

If you have fluorescein (10-100nM) in your lab, you could measure the effect of varying the amount of glycerol (0 to 100%) on the fluorescence anisotropy of fluorescein in a phosphate buffer. The anisotropy should increase with increasing concentration of glycerol. Fluorescein by itself in a buffer should have very very low anisotropy.

Or you could do a fluorescence quenching experiment by titrating potassium iodide (0 to 500mM KI) into a fluorescein solution. The anisotropy should increase with increasing KI. (because of decrease in fluorescence lifetime)

I have never done the above experiments, as I always had a macromolecular binding assay that worked. But you could give the above recipe a try. It should work.