I am working on identification of a small peptide in brain tissue. We have modified the peptide with alkyne modifications for azide-based clicking. For Click reaction we have used 50 uM CuSO4, 300uM BTTAA, 1.75mM Na-Ascorbate and 5uM Biotin-azide/ 647-Fluorophore-azide/800-Fluorophore-azide. We have tried with different concentrations of azides as well. The problem we are facing is we are getting non-specific click labelling all throughout in all conditions when we are checking it on a gel (20% Tris-Tricine) or in a plate reader.

We have also tried with peptide purification followed by click reaction, that also is giving us non-specific labelling.

Any lead in troubleshooting or explanation would be really helpful.

Thanks