Hi Megha,
my explanation would be that you have a 2:2 complex. Meaning in your
standard assay, you need 2 peptides as dimer to get a dimer of your protein.
Now that you do the competition assay, you probably start with
concentrations, where this 2:2 complex is not saturated yet. Meaning
that your unlabelled peptide forms a dimer with a labelled one and this
induces the 2:2 to form, meaning higher polarisation. At some point,
where your 2:2 is saturated, you start actually competing and
polarisation drops.
I would use another method to investigate the actual stoichiometry.
Also, thinking about your starting concentrations might be important.
Are you sure that your labelled-peptide - protein starting
concentrations are high enough above Kd that you have 99% complex formation?
Best,
Raphael
Am 21.06.17 um 21:20 schrieb megha abbey:
Hello All,
This is an off-topic question. I have some issues regarding
Fluorescence Polarization competitive displacement assay and would
need some advice.
I have developed an in vitro fluorescence polarization based assay
using a N-terminus labelled FITC peptide. The peptide is 21 amino
acids long and the binding protein is 50 Kda in size. The Kd for
interaction is 1 uM. I am further running a competitive displacement
assay using the exactly same unlabelled peptide. Here, with increasing
concentration of the unlabeled peptide, the polarization signal first
increases and then shows a sharp decline. Attached is the raw data
file for the above.
I believe that if the unlabelled peptide had been aggregating, the
polarization signal would increase but not drop. If the binding would
have been non-specific, then the unlabelled peptide should not
displace at all, but here I see an increase followed by a decrease in
the signal. What does this increase and sharp drop in polarization
signify and how do I fix this? Please help.
I have checked the polarization for titration of the unlabelled
peptide mixed with fixed conc. of FITC-peptide (no protein added).
Here, the polarization signals are the same for the entire range of
unlabeled peptide. I have also tried incubating the unlabelled peptide
with the protein (for ~15min) first followed by addition of
FITC-peptide, but the results are the same.
Thank you,
Megha
--
Dr. Raphael Gasper-Schönenbrücher
Manager of X-ray Crystallography and Biophysics Unit
Max-Planck-Institute of Molecular Physiology
Room A2.23
Otto-Hahn-Str. 11
44227 Dortmund, Germany
Tel +49 (0)231-133 2111
