Cy5 disulfo NHS modification is a post synthesis conjugation to a primary amino group thus an additional modification with an amino group is required. A C6 or C12 amino group can be placed at the 5' or for the 3' end a C3 or C7 amino and for internal positions an amino modified base is used, e.g Amino dT C6.
The diSulfo– Cy5 NHS Ester is a hydrophilic version of Cy5 due to the two sulfo groups. This version is particularly helpful when the standard hydrophobic Cy5 version is not appropriate for the desired application. All NHS ester derivative modifications are post synthesis for oligos and requires a primary amino group on the oligo for conjugation. The amino group can be placed at either the 5’ or 3’ ends an internally as well.
Cy5 can be used as a replacement for Alexa Fluor 647 Succinimidyl Ester, DyLight 650 NHS Ester, Colorada 645 XT A - NHS ester, Fluorescentred 647 reactive, CF647 uccinimidyl ester and PromoFluor ® -647, NHS ester for the required applications.
Reaction scheme for primary amine labelled oligos with NHS ester is shown in the figure below.