I am studying protein-peptide interaction using fluorescence polarization. The peptides are TMR tagged at N-terminal. When I incubate a range of unlabeled protein concentration (0-2000nM) to the fixed concentration of peptide (10nM or 1 nM) in assay buffer ( Hepes and NaCl), The FP values observed are not following a trend either decrease of increase in case of interaction, and almost similar in case of low or no interaction. Rather the values are jumbled, not similar in replicates well also. The machine is working fine as I am doing protein-protein interaction study on regular basis.
Positive comments re welcome!
Is the fluorescence intensity of the peptide high enough? How does it compare to the background without peptide?
Is the peptide sticking to the plate or cuvette surface? Have you included some detergent to prevent this?
Is the protein aggregated and causing light scattering? Check by DLS or SEC-MALS.
Hi Shanti, are you using a UV/Vis Circular dichroism (CD) spec?
Yes ! the intensity is very good approx 26000 in only labeled peptide well and the peptide-protein well as well. I am not sure if the peptide is sticking to the surface of the plate, though I am using non binding 96 well plates and I have not added detergent also. How much and which detergent is recommended ?
I often use 0.01% Triton X-100. But be careful not to use old, oxidized Triton. Use a fresh bottle or, better still, buy it in ampules (Thermo Scientific SurfactAmps-100).
Hello Adam, I have added 0.01% Triton and 4% DMSO also but still there is significant variability. The mP values should not fluctuate in replicates significantly. I have attached the data file for you reference. The black are protein-peptide mix and red is peptide only. The conc of unlabeled protein is deceasing from top to bottom but conc of labeled peptide is fixed.
Do you have measurements from wells without the peptide? What is the fluorescence intensity?
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