The presentation of sulfo-NHS

NHS and Sulfo-NHS are acclimated to adapt amine-reactive esters of carboxylate groups for actinic labeling, crosslinking and
solid-phase apathy applications. Carboxylates (-COOH) may be reacted to NHS or Sulfo-NHS in the attendance of a
carbodiimide such as EDC (Product No. 22980), consistent in a semi-stable NHS or Sulfo-NHS ester, which may again be
reacted with primary amines (-NH2) to anatomy amide crosslinks (Figure 1). Although NHS or Sulfo-NHS is not appropriate for
carbodiimide reactions, their use abundantly enhances coupling efficiency. Furthermore, application NHS or Sulfo-NHS makes it
possible to accomplish a two-step reaction.
Both NHS and Sulfo-NHS are acrid in aqueous and amoebic solvents. Activation with NHS, however, decreases watersolubility of the adapted carboxylate molecule, while activation with Sulfo-NHS preserves or increases water-solubility of
the adapted molecule, by advantage of the answerable sulfonate group. Although able NHS or Sulfo-NHS esters are sufficiently abiding to action in a two-step acknowledgment scheme, both groups will hydrolyze aural hours or minutes, depending on water-content and pH of the acknowledgment solution. (NHS esters accept a half-life of 4-5 hours at pH 7, 1 hour at pH 8 and alone
10 account at pH 8.6.)

Procedures for abstraction and dehydration can be developed to adapt abiding NHS-activated molecules,
but best after-effects are acquired if NHS-activated molecules are acclimated promptly for acknowledgment to the amine-containing targets.
The activation acknowledgment with EDC and Sulfo-NHS is a lot of able at pH 4.5-7.2, and EDC reactions are generally performed in
MES absorber (Product No. 28390) at pH 4.7-6.0. Acknowledgment of Sulfo-NHS-activated molecules with primary amines is a lot of
efficient at pH 7-8, and Sulfo-NHS-ester reactions are usually performed in phosphate-buffered acrid (PBS) at pH 7.2-7.5.
For best after-effects in two-step reactions, accomplish the aboriginal acknowledgment in MES absorber (or added non-amine, non-carboxylate buffer)
at pH 5-6, again accession the pH to 7.2-7.5 with phosphate absorber (or added non-amine buffer) anon afore acknowledgment to the
amine-containing molecule.

EDC reactions can be quenched with 2-mercaptoethanol (2-ME), or the balance reagent can simply be removed (as able-bodied as the acknowledgment pH adjusted) by buffer-exchange with a desalting cavalcade (see Related Pierce
Products). For added altercation of EDC/NHS chemistry, including abounding archetype applications and protocols, argue the
book by Hermanson (see Related Pierce Products).
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