I have a peptide X which was identified by phage display to be able to bind to a immune cell type specifically with high affinity. To assess the binding by flow cytometry, I stained the cells with antibodies for the surface markers together with the biotinylated peptides (X or the scrambled form of X) in one step and then did a secondary staining with streptavidin-FITC after washing. The flow result was weird: The average fluorescent intensity of one surface marker was significantly reduced in the peptide X treated group compared to other groups. Also, the scrambled X treated group also showed a lot of binding indicated by FITC fluorescence. Could anyone tell me how to interpret this and avoid this peptide-antibody interaction?
Is it possible that the peptide is binding the cells through the same target as your staining antibody? In that case, they would compete...This is just a possibility to think about. If you have information to exclude that, then you would have to think in other technical details.
Thanks Gertrudis! I do have information to exclude possibility of competition of binding to the same targets. What technical issue could be possible?
Is the peptide expected to be have any biological efefct on the cells. Could it be inducing downmodulation of some surface markers due to activation or any other physiological change?
For the apparent high binding of the scrambled peptide, there are three possibilities:
1) It actually is binding.
2) The peptide isn't binding but the streptavidin-FITC is.
3) None of it is binding, you just have poor compensation and there is bleed over from another channel.
To address #3: Do you have a -1 control where you have all your stains in there except the peptide/scramble/streptavidin-FITC? This should ensure correct compensation (the sample should be negative for FITC...or help you define where to set "negative").
To address #2: Do you have a control where you don't have the peptide/scramble but you do have the streptavidin-FITC? This should also be negative (no higher than your unstained control).
To address #1: I'm not sure what kind of cells you are targeting or what receptor on the cells, but do you know specifically how the peptide binds, and did you design the scramble accordingly? For example, peptides in an MHC groove often bind on the outer edges, so scrambling the middle would have no effect. Maybe instead of a scramble you should make a completely irrelevant peptide?
As for your peptide reducing staining of a different receptor, I have to agree with Gertrudis. It sounds like the peptide/biotin/streptavidin/FITC complex is somehow inhibiting binding. Do the two receptors cluster? Even if the peptide doesn't bind the second receptor directly, it could sterically hinder access if the two receptors are close enough.
Are these cells fixed or live?
Thanks Gertrudis and Julie,
The binding target of the peptide was experimental determined to be some protein other than the receptor whose staining was reduced, also not MHC. However, I do not know whether there's a biological effect of the binding. I have both of the controls that Julie mentioned. Both of the controls showed significant less binding than my peptide-SA-FITC staining samples.
I guess Julie's probably right. My scrambled peptide is a random scrambled version of my peptide, which could be somewhat binding or even possibly binding to other more prevalent target on the cells. I should instead use some irrelevant peptides.
Thanks again!
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