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5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *Mixed isomers* FITC-NHS

5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *Mixed isomers* FITC-NHS

Fluorescent Dye Carboxylic Acids and Their Succinimidyl Esters?? ?Succinimidyl esters are proven to be the best reagents for amine modifications because the amide bonds that are formed are essentially identical to, and as stable as the natural peptide bonds. These reagents are generally stable and show good reactivity and selectivity with aliphatic amines.??There are few factors that need be considered when SE compounds are used for conjugation reaction:?1).?Solvents:For the most part, re...

Fluorescent Dye Carboxylic Acids and Their Succinimidyl Esters

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? ?Succinimidyl esters are proven to be the best reagents for amine modifications because the amide bonds that are formed are essentially identical to, and as stable as the natural peptide bonds. These reagents are generally stable and show good reactivity and selectivity with aliphatic amines.??There are few factors that need be considered when SE compounds are used for conjugation reaction:?

1).?Solvents:For the most part, reactive dyes are hydrophobic molecules and should be dissolved in anhydrous dimethylformamide (DMF) or dimethylsulfoxide (DMSO).?

2).?Reaction pH:The labeling reactions of amines with succinimidyl esters are strongly pH dependent. Amine-reactive reagents react with non-protonated aliphatic amine groups, including the terminal amines of proteins and the?e-amino groups of lysines. Thus amine acylation reactions are usually carried out above pH 7.5. Protein modifications by succinimidyl esters can typically be done at pH 7.5-8.5, whereas isothiocyanates may require a pH 9.0-10.0 for optimal conjugations.?

3).Reaction Buffers:Buffers that contain free amines such as Tris and glycine and thiol compounds must be avoided when using an amine-reactive reagent. Ammonium salts (such as ammonium sulfate and ammonium acetate) that are widely used for protein precipitation must also be removed (such as viadinlysis) before performing dye conjugations.?

4).?Reaction Temperature:Most conjugations are done at room temperature. However, either elevated or reduced temperature may be required for a particular labeling reaction.


5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester]

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Features and Biological Applications

5(6)-FAM, SE is the amine-reactive succinimidyl ester of FAM acid. It is favored over FITC in bioconjugations. FAM reagents give carboxamides that are more resistant to hydrolysis. In addition, FAM reagents require less stringent conjugation conditions and give better conjugation yields, and the resulted conjugates have superior stability. FITC-labeled nucleotides and peptides tend to deteriorate more quickly than the corresponding FAM conjugates. We found that FAM reagents can be used to substitute FITC reagents in most biological applications.
References

1. ? Hahn M,?et al. (2001).??Influence of fluorophore dye labels on the migration behavior of polymerase chain reaction-amplified short tandem repeats during denaturing capillary electrophoresis.?Electrophoresis22, 2691-700.

2. ? Sanders SJ (2000). Factor V Leiden genotyping using real-time fluorescent polymerase chain reaction.?Mol Cell Probes14,?249-53.

3. ?Brandis JW (1999). Dye structure affects Taq DNA polymerase terminator selectivity.?Nucleic Acids Res27,?1912-8.

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