Usually FPIA is used for small molecules. The larger the analyte, the smaller the difference in the bound and the free form.

https://en.wikipedia.org/wiki/Fluorescence_polarization_immunoassay

Hello Michael, and thank you for your observation! I know FPA is ideally used for small molecules, however FPIA have been set up for antigens with MW similar or even higher tan that of my protein (flagellin protein from Leptospira of 31kDA, polysaccharide from Brucella of 22 kDa, MBP from M bovis of 18 to 23kDa...)..

Yes, you are right. However, to my knowledge, you need special labels (with long decay times) for these applications. Could you supply links to the respective articles?

Here are the links to the papers I have refered to!

http://cvi.asm.org/content/3/4/438.long

http://www.sciencedirect.com/science/article/pii/S0378113502000445?via%3Dihub

Obviously it is possible to perform FPA with antigens around 20kDa. When I checked your protocol, I was surprised about the high excess of FITC. If too many fluorphores are attached, self-quenching will occur, which might lead to strange effects. Did you check the conjugation density? About 1-2 FITC molecules per protein should be optimal. Please also consider that a high FITC load might destroy most of the epitopes, which are bound by the antibodies.

I return back to this task!!. Thank you for your answer again!!

I also think it is an excess of FITC... I did not checked the conjugation density, as I do not know exactly de extinction coefficient of my protein. Further, after some experiments I realized my protein was begining to break down, even when it was a -20°C. The antigenic properties of the protein are still OK after coupling to FITC (checked by dot and western blott).

I will make a new conjugation , adding a protease inhibitor after FITC is removed. Do you recommend me any suitable FITC/ protein relatinship, or should I have to test? (I have limited quantities of the protein!!) Is it recommended to add glycerol to the conjugate in order to preserve it at -20°C?

to increase protein stability, better add a preservative like sodium azide to prevent microbial growth and (if the assay permits) some unlabeled protein like BSA to a concentration of 5..10mg/ml, instead of costly protease inhibitors and try storage in the fridge, not in the freezer. If you suspect protease contamination in your protein prep, use a SEC column (e.g. Sephacryl S200 for your target MW range) instead of the Sephadex G25 for cleanup (the latter has a sharp cut-off at about 10kDa and just is removing unreacted fluorochrome). Contaminating proteins easily should be detectable by fluorescence, as they also will be labeled.