What are NHS esters?
What are NHS esters?
NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules (see Carbodiimide Crosslinker Chemistry). NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds.
What does NHS ester react with?
-amines
In addition to the expected reactivity with N-terminal α-amines and lysine side-chain ε-amines, NHS esters can react and couple with tyrosine, serine, and threonine –OH groups.
How do you quench NHS esters?
NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate/bicarbonate, HEPES and borate buffers. Other buffers may also be used, provided they do not contain primary amines such as Tris or glycine. Using a large excess of Tris or glycine at neutral-to-basic pH can quench the reaction.
What is EDC NHS?
EDC couples NHS to carboxyls, forming an NHS ester that is considerably more stable than the O-acylisourea intermediate while allowing for efficient conjugation to primary amines at physiologic pH. Sulfo-NHS plus EDC (carbodiimide) crosslinking reaction scheme.
How stable is NHS ester?
In relation to other active compounds, NHS esters are relatively stable and can be stored for months under dry conditions. However, in practice, hydrolysis of NHS esters is a frequent reason for suboptimal conjugation results or even a complete failure of the respective derivatization reactions.
Does NHS ester react with alcohol?
NHS esters have negligible reactivity with alcohol [15].
Does NHS ester react with secondary amine?
It works with secondary amines as well.
How do I make an EDC solution to the NHS?
Protocol
- Equilibrate the EDC and NHS to room temperature before opening.
- Prepare 1ml of a 1mg/ml solution of Protein #1 in Conjugation Buffer 1.
- Add 0.4mg EDC and 0.6mg NHS or 1.1.mg sulfo-NHS and react for 15 minutes at room temperature.
- Add 1.2µl 2-mercaptoethanol to quench the EDC.
How do I prepare for EDC NHS?
What is EDC reagent?
EDC hydrochloride is a coupling reagent. Classified as a carbodiimide, EDC hydrochloride is carboxyl activating and water soluble. EDC hydrochloride is used for amide bond formation in oligonucleotides and tetraazamacrocycles.
How do you store sulfo-NHS?
NHS and Sulfo-NHS are relatively stable in solution but best results are obtained when they are dissolved immediately before use. Store these compounds desiccated at 4°C.
Does arginine react with NHS ester?
Thus, as has also been noted for primary amines in the gas-phase,13 protonation of the arginine side-chain renders it non-nucleophilic and unreactive towards NHS esters.
