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This is intended as a guide only for protein/antibody labeling; optimal conjugation protocols may vary depending on the target being labeled.
Succinimidyl esters (NHS esters) are amine-reactive reagents that can be conjugated to (non-protonated) aliphatic amine groups. The primary reactive species for protein amine-conjugation are the ε-amino groups of lysine residues. To avoid protonating these groups it is important to perform the reaction at a slightly basic pH. In addition, buffers containing primary amines should be avoided, since they will compete for conjugation with the NHS ester.
Please note that NHS esters can be moisture sensitive, so handle accordingly. Where possible, handle and store fluorescent dyes in the dark.
Note: A typical molar ratio for labeling Janelia Fluor®, SE dye:protein is 15:1, however this should be optimized for each specific protein. We recommend trying three different molar ratios of dye:protein to develop a protein-specific protocol for future use.
† N.B. This is critical for achieving the optimal degree of labeling (DOLs) for Janelia Fluor® dyes.
† N.B. Increasing the incubation time to 18 hours in the dark can increase the DOL with Janelia Fluor® dyes.
A280c = A280 - (Amax x CF)
Please refer to the individual product descriptions for the correction factor (CF) values for the fluorescent dye used.
A280 = ε x [protein] x l
|F:P =||Amax x MWprotein|
|[protein] x εdye|
Zeba is a trademark of Thermo Scientific and MiniTrap is a trademark of GE Healthcare.