Hi,
I performed a competition assay of serially diluted concentrations of the unlabeled peptide by adding a constant concentration of the FITC-labeled peptide. I have observed higher fluorescence intensity in higher concentrations of unlabeled peptide compared to the lower concentrations of the unlabeled peptide. As per literature we should have lower intensity in higher concentration so of unlabeled peptide. Could you kindly clarify why this is happening or should it be the opposite.
Dear Hanni Grace F You've provided obviously too little detail to see what is really going on but have a look at the following paper: Article Time-resolved FRET and NMR analyses reveal selective binding...
(see enclosed file for the final edited version) and have a close look at Figure 2. Here you see that frequently higher values are found in the lower ranger.Based on the limited info you provided a possibility might be that you have to use higher concentrations of the unlabelled peptide (in the range of 100 or 1000 uM) to see a decrease.
Best regards.
Dear Keller,
I have gone through the paper you had sent. In this paper FRET has been used to analyze the competition assay. The experiment which I did was done using flow cytometry, we have done this experiment three times and yet we aren't getting any competitive effect by using labelled-FITC (10uM constant concentration) and unlabeled peptide 100uM to 1.25uM concentration . The results in many previous reported literature for competition assay have used spectrophotometer for analysis, but in FACS analysis the incubation time is too much (24hours) which is not good for my cells. Could you please clarify more on that, also if you have any suggestion for competition assay for flow cytometry analysis kindly let me know. Also as you have suggested and according to the paper you sent I will try using log concentrations of the unlabeled peptide (0.0001 to 1000um).
Best Regards,
Hanni
Article Competition-based cellular peptide binding assays for 13 pre...
Hi , I have used the protocol mentioned in this paper.
Dear Hanni Grace F ,
Thanks for the additional info. I am afraid that it is impossible to pinpoint the exact reason for your observation since there are too many steps in the described method that might be of influence. All I can suggest is:
-Use (if possible) a control peptide to see if your approach works and renders results as published before. In the ideal world you must find reproducible results
-Perhaps lowering the labeled peptide concentration to 5 uM instead of 10 might increase the affinity specificity
-Out of my own experience I know that the FITC fluorescence and/or absorbance is extremely sensitive for things like temperature, ionic strength and pH. In other words, be absolutely sure that these factors are the same for every dilution experiment (if you add for example 10uL of concentrated unlabeled peptide solution then add 10uL of a less concentrated unlabeled peptide solution as well and not for example 1 uL)
-‘Play’ around with the sequence of events, add unlabeled peptide first and then labeled peptide, the other way around and perhaps even try a mixed solution so you can add them (labeled and unlabeled) at the same time
I am sorry that I cannot be more concrete, but the protocol you use is so labor-intensive and involves so many different steps that may, or may not, have an influence that it is difficult if not close to impossible to find a 1-on-1 cause for your observation.
Best regards.
PS. Although a highly unlikely scenario…one thing crossed my mind. The protocol is in my view somewhat unclear when it comes to concentrations. I mean a 5 uM peptide concentration for example does this means the peptide concentration of the solution from what you add if they indicate let’s say 5 uL or does 5 uM means the end-concentration of the peptide in the total volume of the assay used.
Anyway, what crossed my mind is that FITC is highly sensitive for self-quenching. Suppose that the FITC labelled peptide ‘aggregates’ at the cell surface, so highly clustered, then adding unlabeled peptide ‘dilute’ the fluorescence label and this results in a higher intensity and if something like this happened it could explain your observation.
Though not entirely the same, the principle of adding unlabeled peptide to ‘eliminate’ the self-quenching was used here: Article Tryptophan fluorescence study on the interaction of the sign...
See also Figure 8.5 in another study, with just another illustration of how sensitive FITC is for self-quenching etc.: Chapter Characterization of helical junction zones in gelatin networ...
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