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>I have recently conjugated 2 mAbs to Fluorescein succinimidyl ester (SFX),
>marketed as a more stable fluorescein conjugate and sold by Molecular
>Probes. The conjugations worked well and give good signal to noise, however
>they seem to require significantly more compensation than my FITC mAbs. In
>specific my FL2-%FL1 typically runs w25% with commercial FITC labelled mAbs
>but runs w45% with FSX. Molecular Probes technical service claims the
>fluorescence emisson spectra of FITC and FSX are identical, however my
>compensation data would suggest otherwise. Has anyone else out there had
>any experience with this fluorophore in regards to compensation?
We've tried it here and have had similar results. Therefore we continue to use
FITC in our products. However, we didn't require quite as much extra
compensation as you're reporting. You might want to vary the SFX reaction
ratio's to assess the change in F/P and the consequential effect on compensation
if you still want to use it. If it's overlabeled there can be a red shift.
Regards
Tony Ward
Associate Product Manager
BDIS
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