I try to do some displacement assay, i.e. a fluorescence labeled receptor ligand should be displaced by an unlabeled one in a cell culture binding study. I used the unlabeled one in 1000 times higher concentration, but instead of measuring a diminished fluorescence signal the signal was higher.
The problem could be because you are not using purified components. There are several issues. First, does the fluorophore increase in signal with just the unlabeled ligand and no receptor. Second, there is fluorescence due to the cells, that can be affected by the fluorophore. Also, in a cell assay, there are many " receptor" type proteins. Perhaps there is an abundant protein that binds the either the fluorophore or the unlabeled ligand better.
Given all the problems you may have with a cellular assay, typically, the fluorescence of the labeled ligand can go either up or down when it is displaced. So it would not be surprising to me if the fluorescence goes up instead of down.
The problem could be because you are not using purified components. There are several issues. First, does the fluorophore increase in signal with just the unlabeled ligand and no receptor. Second, there is fluorescence due to the cells, that can be affected by the fluorophore. Also, in a cell assay, there are many " receptor" type proteins. Perhaps there is an abundant protein that binds the either the fluorophore or the unlabeled ligand better.
Given all the problems you may have with a cellular assay, typically, the fluorescence of the labeled ligand can go either up or down when it is displaced. So it would not be surprising to me if the fluorescence goes up instead of down.
Dear Marcia,
You can always try a label free method. Check out the attached paper
Article Talking Sense: Cell based biosensors
Dear Marcia
I too performed similar experiments. I am not sure about your competition assay protocol so I will just discuss my results.. I performed Sequential (First 2 h unlabeled then wash and incubate with labeled peptides) and simultaneous competition (incubation of cells with with labeled and unlabeled peptide together for 2 h). Interestingly the results of Simultaneous assay showed 80% reduction in signal while the sequential competition effects were minor. At the moment I am not sure about the reason but in my opinion it is due to the receptor mediated endocytosis and recycling of receptors. I discussed with my colleagues and some of them said it might be due to weak affinity of the peptides means washing in sequential competition might remove the unlabeled peptide. However I give same washing each time I add labeled peptide and it gives strong signal. Therefore for me affinity does not seem to be a reason.
Hope it helps...
Thank a lot for your answers.
@Teodor Aastrup, the paper for the label free method, where can I find it?
@ Marcia Moss: How can I purify the fluorecence labeled ligand? The problem is that this component is 1979 Da and the fluorophore is 1620 Da. So clean up by centrifugation and microfiltration seems to be not so easy.
@ Prafull Singh, I used a combination of both. 10 min preincubation with the label free and than adding the labeled receptor ligand. The overall incubation time was 1h.
Dear Prafull,
Labeling can affect the binding properties, both directly both also indirectly by introducing off-target interactions.The latter can be obesterved in cell-based assays if you have good time resolution and avoid washing steps. The effect of the labels on the binding can also affect the kinetics, normally the association phase. It is usually more pronounced in cell-based assays compared to biochemical assays, because in the biochemical assays one has an artificially high accessibility of the receptors.
Feel free to contact me if you want to discuss this more.
Best regards
Teodor
If you have suitable instrumentation at hand (many plate readers do), you could use fluorescence polarization in addition to fluorescence intensity to monitor the binding process. Fluorescence polarization does not depend on the actual fluorescence intensity and should be notably increased upon binding of the labelled ligand by the receptor.
- Badges
- Science topic