Tyramine is a primary amino compound obtained by formal decarboxylation of the amino acid tyrosine:
Below is the protocol for labeling of tyramine molecule with Cyanine3 NHS ester product for use in tyramide signal amplification.
All steps protected from light and performed at room temperature.
Solution 1: Cyanine 3 NHS ester 10 mg/ml in Dimethylformamide (DMF) (1 mg in 100 ul)Solution 2: 1% Triethylamine (TEA) in DMF (10 ul TEA in 1 ml DMF)Solution 3: 10 mg/ml Tyramine HCl in Solution 2 (10 mg Tyramine HCL in 1 ml Solution2)
Reaction: Mix together 100 ul of Solution 1 + 27 ul of Solution 3; incubate for 4 hours at room temp, then add 1.2 ml 100% EtOH.
Results tested side by side with known working fluorescent tyramide conjugates via FISH with digoxigenin labeled RNA probes and HRP-conjugated anti-digoxigenin antibodies.
The Cy3-labeled tyramide was tested at several concentrations and showed no staining whatsoever while control slides showed normal staining. This same reaction has been carried out successfully in the past with Lumiprobe CyanineNHS ester dyes as well as FAM NHS ester and TAMRA NHS ester.