I am trying to conjugate 5/6 Fam-NHS ester to a peptide in solution. I do see a conjugated product that has a mass of my peptide +3 units of 5/6 Fam but that mass is also exact to the 9 subunits of 5/6 fam itself?
Has anyone observed that too? I know I can do MS/MS on the peak to know if it certainly is my peptide with fam units or a multimer unit of Fam?
But just as a curiosity I would like to know if that has been ever observed that before?
inputs will be much appreciated.
Does the peptide have at least 3 primary amino groups (N-terminus and Lysine) to account for the 3:1 stoichiometry? If so, that is probably what you made.
I have never heard of self-reaction of the reagent being a problem. The dye has no amino groups to react with NHS ester.
By the way, you may find that having 3 FAMs/peptide is too many. The peptide may become very hydrophobic, and the dye molecules may quench each other's fluorescence.
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