Amine functional group free#

In addition, the charged group prevents Sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods. This charged group has no effect on the reaction chemistry, but it does tend to increase the water-solubility of crosslinkers containing them.

Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. Impure glycerol and high concentrations (20-50%) of glycerol also decrease reaction efficiency. Low concentrations of sodium azide (≤ 3 mM or 0.02%) or thimerosal (≤ 0.02 mM or 0.01%) generally do not significantly interfere with NHS-ester reactions, but higher concentrations do interfere.

Primary amine buffers such as Tris (TBS) are not compatible, because they compete for reaction however, in some procedures, it is useful to add Tris or glycine buffer at the end of a conjugation procedure to quench (stop) the reaction. NHS-ester crosslinking reactions are most commonly performed in phosphate, carbonate-bicarbonate, HEPES or borate buffers at pH 7.2 to 8.5 for 0.5 to 4 h at room temperature or 4☌.

Amine functional group free#

The extent of NHS-ester hydrolysis in aqueous solutions free of primary amines can be measured at 260 to 280 nm, because the NHS byproduct absorbs in that range. This half-life decreases to 10 mins at pH 8.6 and 4☌. The half-life of hydrolysis for NHS-ester compounds is 4 to 5 h at pH 7.0 and 0☌. The rate of hydrolysis increases with buffer pH and contributes to less-efficient crosslinking in less-concentrated protein solutions. Hydrolysis of the NHS ester competes with the primary amine reaction. The functional groups are frequently used in protein biology methods. However, NHS esters and imidoesters are the most popular amine-specific functional groups that are incorporated into reagents for protein crosslinking and labeling. The isothiocyanate group is familiar to researchers who have used the traditional fluorescent labeling reagent called FITC (fluorescein isothiocyanate). These compounds are used to fix and preserve tissues or cells for immunohistochemistry (IHC) applications. Most of these conjugate to amines by either acylation or alkylation.įormaldehyde and glutaraldehyde are aggressive carbonyl (–CHO) reagents that condense amines via Mannich reactions and/or reductive amination. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. In fact, there are numerous synthetic chemical groups that will form chemical bonds with primary amines. Furthermore, among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic this makes them easy to target for conjugation with several reactive groups. These primary amines are positively charged at physiologic pH therefore, they occur predominantly on the outside surfaces of native protein tertiary structures where they are readily accessible to conjugation reagents introduced into the aqueous medium. Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys, K) amino acid residues. The simplest, most common and versatile techniques for crosslinking or labeling peptides and proteins such as antibodies involve the use of chemical groups that react with primary amines (–NH2).