Hi guys,

I am trying to develop a Fluorescence polarization based microplate assay for analyzing protein-nucleic acid binding. So I have fluorescein labelled DNA (short oligos betwen 14 – 50 bps)that I titrate with different concentrations of proteins and then look for a saturation in anisotropy.

It is definitely a straightforward assay…but my problem is that I am not convinced with the Intensity values that my microplate reader churns out for the 1nM fluorescein labelled oligo that I have.

So my question is

“How do I make sure that I am within the acceptable working range of fluorescence intensity when I do my assays with the fluorescein labelled oligos”?

Though it appears to be quite a simple question, I am kind of bugged with it.

The user manual for the microplate reader Spectramax M5) does not have any standard spectra or the fluorophore concentration Vs intensity plots that I can refer to.

I know that there are some specialists here :)

Any inputs ?