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Amine-reactive crosslinker reactive groups



Formaldehyde and glutaraldehyde are aggressive carbonyl (–CHO) reagents that condense amines via Mannich reactions and/or reductive amination. These compounds are used to fix and preserve tissues or cells for immunohistochemistry (IHC) applications. The isothiocyanate group is familiar to researchers who have used the traditional fluorescent labeling reagent called FITC (fluorescein isothiocyanate).


Selected amine-reactive chemical groups. The functional groups are frequently used in protein biology methods.

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Crosslinking Reagents Technical Handbook


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N-hydroxysuccinimide esters (NHS esters)

NHS ester reaction chemistry


NHS ester reaction scheme for chemical conjugation to a primary amine. R represents a labeling reagent or one end of a crosslinker having the NHS ester reactive group; P represents a protein or other molecule that contains the target functional group (i.e., primary amine).

Hydrolysis of the NHS ester competes with the primary amine reaction. The rate of hydrolysis increases with buffer pH and contributes to less-efficient crosslinking in less-concentrated protein solutions. The half-life of hydrolysis for NHS-ester compounds is 4 to 5 h at pH 7.0 and 0°C. This half-life decreases to 10 mins at pH 8.6 and 4°C. 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.


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. Impure glycerol and high concentrations (20-50%) of glycerol also decrease reaction efficiency.

Sulfo-NHS esters are identical to NHS esters except that they contain a sulfonate (–SO3) group on the N-hydroxysuccinimide ring. This charged group has no effect on the reaction chemistry, but it does tend to increase the water-solubilty of crosslinkers containing them. In addition, the charged group prevents Sulfo-NHS crosslinkers from permeating cell membranes, enabling them to be used for cell surface crosslinking methods.




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  • Tech Tip #3: Determine Reactivity of NHS Ester Biotinylation and Crosslinking Reagents

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Bioconjugate Techniques, 3rd Edition



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Applications for NHS ester crosslinking

1. Protein interaction analysis




Comparison of several in vivo crosslinking methods. HeLa cells treated with 1% Formaldehyde (HCHO) or 1 mM homobifunctional NHS-ester crosslinker (Thermo Scientific DSG and DSS) in PBS for 10 mins before quenching. A fourth set of HeLa cells were treated and crosslinked for 10 mins with 4 mM  Photo-Leucine, 2 mM  Photo-Methionine (Photo-AA) according to the procedure (see subsequent section). Formaldehyde-treated and NHS-ester-treated cells were quenched with 100 mM glycine pH 3 and 500 mM Tris pH 8.0, respectively for an additional 15 mins. One million cells from each condition were then lysed and 10 µg of each sample was heated at 65°C for 10 mins in reducing sample buffer containing 50 mM DTT and analyzed by SDS-PAGE and western blotting with Stat3 specific antibodies (Cell Signaling). GAPDH (Santa Cruz) and beta-actin (US Biologicals) were blotted as loading controls.

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2. Prepare specific protein conjugates




High levels of maleimide activation of Thermo Scientific Imject Carrier Proteins. Surface primary amines (i.e., lysine side chains) of Imject Carrier Proteins were maleimide activated by reaction with excess Thermo Scientific Pierce Sulfo-SMCC crosslinker. Activation levels (moles of maleimide per gram of carrier protein) were determined with a cysteine coupling assay. KLH and Blue Carrier Proteins are very large (approx. 8000 kDa), enabling activation with 600 to 900 maleimide groups per protein molecule. BSA and ovalbumin are smaller (67 kDa and 45 kDa, respectively), allowing for activation with 5 to 20 maleimide groups per protein molecule.

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3. Label antibodies and other proteins



To generate the he fluorescent western blot data that follows, a fluorophore conjugated secondary antibody was used to detect the target protein, ERK1 in cancer cell lysate. 

Infrared western blot detection of ERK1 using DyLight 755-conjugated secondary antibody. Serial dilutions of A562 cell lysates were analyzed by western blotting for levels of ERK1 using a mouse anti-ERK1 primary antibody and a goat anti-mouse secondary antibody conjugated to DyLight 755 (using the DyLight 755 Microscale Antibody Labeling Kit). Left lane: Infrared molecular weight marker.

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4. Immobilize antibodies and other proteins



In addition, homobifunctional NHS crosslinkers like DSS, described above, are frequently used to retain an antibody on the beads during immunoprecipitation (IP) procedures. This "Crosslink IP" method (also called IgG Orientation) involves first binding the purified IP-antibody to the Protein A/G agarose resin, then adding DSS to covalently crosslink the affinity-bound antibody and Protein A/G molecules through respective primary amines. This representative protein gel provides an example of results generated using the crosslink IP method. 


Specific protein purification using IgG coupled to NHS-activated agarose. Human IgG (25 mg) was immobilized on 1mL of Pierce NHS-Activated Agarose. The affinity resin was used to purify secreted recombinant Protein G from E. coli. A final yield of 25 mg of protein was obtained. Lane 1: bacterial pellet, Lane 2: MW marker, Lane 3: culture supernatant, Lane 4: flow-through, Lanes 5-12: elutions, Lane 13: boiled resin following elutions.

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Imidoester reaction chemistry


Imidoester reaction scheme for chemical conjugation to a primary amine. R represents a labeling reagent or one end of a crosslinker having the imidoester reactive group; P represents a protein or other molecule that contains the target functional group (i.e., primary amine, –NH2).

Applications for imidoester crosslinking



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Recommended reading
  1. A practical approach to crosslinking Mattson, G., et al. Molecular Biology Reports (1993) 17:167-183.
  2. Grabarek, Z. and Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Anal Biochem 185:131-5.
  3. Staros, J.V., et al. (1986). Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 156:220-2.
  4. Timkovich, R. (1977). Detection of the stable addition of carbodiimide to proteins. Anal Biochem 79:135-43.