Super Fluor 488，555, 647, 680, 750 NHS Ester (Succinimidyl Ester)

Super Fluor® is registered trademarks and strictly forbidden to be used without authorization.

     Super Fluor^® dyes are the ideal substitute for Alexa Fluor dyes. Super Flour 488 is a bright and photostable dye with the same chemical structure of Alexa Fluor 488 for demanding applications including microscopy. Due to its high hydrophilicity, this is a dye of choice for the labeling of sensitive proteins and antibodies. The dye is useful for many demanding applications, including microscopy. From the chemical standpoint, Super Flour 488 is equivalent of Alexa Fluor 488 with a sulfonated rhodamine dye Rhodamine 110 (R110). Like other rhodamines, it is available as 5- and 6-isomers, which have almost identical photophysical properties. The Super Flour 488 NHS ester is an amine reactive dye, it can label amine groups in proteins, peptides, amino-modified oligos, and other target molecules.

NHS ester labeling of amino biomolecules

Super Fluor 488，555, 647, 680, 750

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: Dissolve NHS ester in 1/10 reaction anhydrous volume of DMF or 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) Use 10:1 molar ratio Dye NHS/ protein to perform the labeling. Calculate required amount of NHS ester and the protein.

6) Add NHS ester solution to the solution of biomolecule, and vortex well. Keep on ice overnight, or at room temperature during at least 4 hours.

7) Purify the conjugation. The dye-protein conjugate purification by using a Sephadex G-25 column. Add PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

8) Determine the optimal dye/protein ratio (optional):Each protein requires distinct dye/protein ratio, which also depends on the properties of dyes. Over labeling of a protein could detrimentally affects its binding affinity while the protein conjugates of low dye/protein ratio gives reduced sensitivity. 

 NOTE: Always wear lab coats, gloves and goggles when working with our products although they are low-risk chemicals for R&D only.