Colleagues, good afternoon! We plan to conjugate antibodies with a fluorescent dyes. One source says that it is possible to conjugate anebodies that already have tag in the form of HRP, for example. Another source says this will be ineffective. How do you do this kind of work? Is the dilution of such a conjugate different from the conjugate with horseradish peroxidase?
If you attach your HRP using the same conjugation chemistry as the fluorophore then yes, it's possible that the HRP conjugation blocks your fluorophore attachment residues. It is best to choose a different chemistry for the two conjugates. For example, you could attach your fluorophore NHS (N-reactive) and attach your HRP using streptavidin (Fc-reactive). That way they go to different places stay out of each other's way.
Sounds interesting to me. Conjugating fluorescent dyes to antibodies already linked to HRP? I have conjugated Cy dye, Alexa dye, etc. to antibodies so far. Eventually, I have used them to develop in vitro diagnostic reagents. My concern in your experiment is the stability of the HRP. In order to conjugate with fluorescent dyes, the HRP-linked antibody needs to be exposed for 1-2 hours at room temperature. Of course, you can cover the light... but the stability of the HRP is a concern. However, the only way to know is to do the experiment. I'd be interested to hear your results.
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