Both 6-FAM (6-carboxyfluorescein, single isomer) and FITC (fluorescein-5,6-isothiocyanate, mixed isomers) are forms of the fluorescent dye fluorescein. FITC is a particular form of reactive species, the isothiocyanate, of the dye. It yields a urea linkage upon reaction with a primary amine. 6-FAM is the preferred reagent for labeling an oligonucleotide. It results in an amide bond when reacted with a primary amine. The chemistry is more robust and better yielding. Furthermore, it has been shown that 6-FAM is less susceptible to photobleaching. 6-FAM and FITC conjugated oligonucleotides have similar spectral properties otherwise.
Is 6-FAM-labeled oligonucleotides freely taken by mammalian cells in culture in the absence of transfection agent ? (e.g. a liposome). In other words is 6-FAM permeable to the cells ?
Dear Carlos,
6-FAM isn’t hydrophobic enough to difuse an oligo across the cell membrane. Some modifications that help with cell uptake are multiple incorporations of spermine, or hydrophobic labels like stearyl and cholesterol. Please contact us if you have any additional questions.
Kindest regards,
Danica
Thanks Danica, actually I was thinking more in terms of if the FAM labeling would be preventing the internalization within the cells of an oligo that would enter by itself. Specifically, my question would be if an aptamer oligonucleotide that is internalized within the cell, if it is labeled with FAM would still be entering, or that FAM would block that internalization. Do you have experience with labeled aptamer said or any suggestion in that direction. Thanks a lot
Carlos
Dear Carlos,
FAM will not block update in cells. In fact, FAM has been used extensively in fluorescent in situ hybridization. One thing to note, FAM is quenched under low pH conditions. If your cellular compartment is acidic (e.g. in lysosomes) you will not be able to observe fluorescence.
Please contact us with any additional questions.
Kindest regards,
Danica