Glycerol probably will increase the baseline fluorescence polarization of the probe by increasing the viscosity of the solution. With only 5% glycerol, the effect will be small. There is an equation that relates polarization to viscosity, called the Perrin equation.

Thanks Adam. I will look into that!

I agree fully with the answer of Adam . However I give you some hints concerning the use of:

- 5(6)-carboxyfluorescein (FAM). Please don 't use acronyms for a question.

Give please infos about the labeling of your peptide with FAM (you have 2 COOH or COOR at the beginning (see Molecular probes). I mean purity and stability of your labeled peptide.

Please start GOOGLE SCHOLAR AND NOT GOOGLE  and give your question in the mask.

you 'll earn 35.100hits

here the link:

https://scholar.google.de/scholar?hl=de&as_sdt=0,5&q=Does+glycerol+affect+fluorescence+polarization+result%3F

PLease read the chapter of the Förster 's book (English translation if avaible). It is very useful for the theory concerning your question.

JRG

Hi Jean,

Thanks many for your reply! I will look into that.

My FP assay doesn't work properly even when I match the glycerol concentration or change buffer. I suspect either there is something wrong with the fluorescent peptide or the labeling has disrupts its ability to interact with my protein.

The FP assay between my protein & the peptide was published before so it should work. Don't know why I didn't see any binding upto 60 uM of protein.

I have proven the binding between my protein & peptide by AlphaScreen (same batch of protein); therefore, the protein is properly folded and can bind to peptide.

I am going to try the FP with non-truncated form of my protein and see if I get better result.

Best,

Lyra

Are you using exactly the same ligand and fluorophore as in the published paper?

Dear Lyra,

Find in attach, 2 papers on: A Study of the Adsorption of Dyes on Bovine Serum Albumin by the Method of Polarization of Fluorescence and on: POLARIZED FLUORESCENCE OF MACROMOLECULES.

You'll find in them: may be increased by placing the dye in a viscous medium such as glycerol (Weigert, 1920 ... DJ

R. LAURENCE fluorescence during adsorption would affect intensity measurements made with

a photocell in ... Effect of pH As the pH in the surrounding solution is de- creased and the ...

Also, you'll find in: These direct effects, if present to a significant extent, ought to affect the dependence of ... from

fluorescein in glycerol, A, = 495 nm; (X) obtained from PRODAN in glycerol, A, = 360 ... Besides

the main transition described above, we also examined the pressure effect on pretransitons ...

Best wishes