Amine-reactive crosslinker reactive groups
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). Primary amines exist at the N-terminus of each polypeptide chain and in the side-chain of lysine (Lys, K) amino acid residues. 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. 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.
In fact, there are numerous synthetic chemical groups that will form chemical bonds with primary amines. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. Most of these conjugate to amines by either acylation or alkylation.
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).
However, NHS esters and imidoesters are the most popular amine-specific functional groups that are incorporated into reagents for protein crosslinking and labeling.
Selected amine-reactive chemical groups. The functional groups are frequently used in protein biology methods.
This 45-page guide is of value to the novice as well as those who have previous experience with crosslinking reagents. It begins with a basic discussion on crosslinking and the reagents that are used. The guide also contains a discussion on various applications where crosslinking has been applied, including the powerful label-transfer technique for identifying or confirming protein interactions. Crosslinking chemistry is addressed in an easy-to-follow format designed to convey the important information you need without getting lost in details. Each Pierce crosslinking reagent is shown along with its structure, molecular weight, spacer arm length and chemical reactivity. The handbook concludes with a list of excellent references on crosslinker use and a glossary of common crosslinking terms.
NHS ester reaction chemistry
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. The reaction releases N-hydroxysuccinimide (NHS).
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.
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°C. 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.
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.
Sulfo-NHS ester reaction scheme for chemical conjugation to a primary amine. R represents a labeling reagent or one end of a crosslinker having the sulfo-NHS ester reactive group; P represents a protein or other molecule that contains the target functional group (i.e., primary amine, –NH2).
The solubility of NHS-ester reagents varies with buffer composition and the physical properties of the remainder of the molecular structure (e.g., spacer arm). Many non-sulfonated forms of NHS-ester reagents are water-insoluble and must be dissolved in a water-miscible organic solvent, such as DMSO and DMF, before they can be added to an aqueous reaction mixture. Thus, crosslinking reactions with the water-insoluble NHS-esters typically require an organic solvent-carryover of 0.5 to 10% final volume in the aqueous reaction.
NHS vs. Sulfo-NHS crosslinkers. Structures of DSS and BS3 (Sulfo-DSS) amine-to-amine crosslinkers. DSS is not directly water-soluble but once dissolved can permeate across cell membranes to crosslink inside cells. BS3 is water-soluble (at usual working concentrations) but, being charged, cannot permeate cell membranes; this confines BS3 crosslinking to the surface of intact cells.
Bioconjugate Techniques, 3rd Edition (2013) by Greg T. Hermanson is a major update to a book that is widely recognized as the definitive reference guide to the field of bioconjugation.
Bioconjugate Techniques is a complete textbook and protocols-manual for life scientists wishing to learn and master the biomolecular crosslinking, labeling, and immobilization techniques that form the basis of many laboratory applications. The book is also an exhaustive and robust reference for researchers looking to develop novel conjugation strategies for entirely new applications. It also contains an extensive introduction to the field of bioconjugation that covers all of the major applications of the technology used in diverse scientific disciplines as well as containing tips for designing the optimal bioconjugate for any purpose.
Applications for NHS ester crosslinking
1. Protein interaction analysis
Homobifunctional crosslinkers that have NHS ester groups at both ends, such as DSS and BS3 shown above, are primarily used in applications where the goal is to covalently (permanently) bond together binding partners in protein complexes. Although conjugation can occur between primary amines of any two (same or different) protein molecules, only those proteins that are nonrandomly closely associated in a binding relationship will become crosslinked with sufficient frequency in the entire population of molecules to detect in analysis. Unless the goal is random polymerization, these amine-to-amine crosslinkers are seldom used to crosslink purified proteins that have no binding relationship.
This technique can be applied to discover or validate a protein interaction or to analyze the conditions in which a known protein interaction occurs. Experiments can be done in vitro (with complex lysates or purified putative interacting proteins) or in vivo (intracellular or cell surface). With proper controls, the relative abundance of conjugates of different size and identity (determined by electrophoresis and staining or Western blotting) will be indicative of specific interactions at the time of crosslinking. By comparing results using crosslinkers with different spacer arm lengths or different cleavability or solubility features, different characteristics of an interaction can be elucidated.
Heterobifunctional NHS-ester crosslinkers, in particular those with opposite ends that contain a photo-activatable group, are particularly useful for protein interaction analysis. These linkers can be reacted first to a purified "bait" protein (via NHS-ester reaction to primary amines) and then added to cells or a lysate to allow the bait protein to interact with the "prey" protein. When desired, the second end of the linker can be activated (with UV-light) so that it attaches to whatever chemical group it first encounters. In a protein interaction complex, the reaction will result in crosslinks between bait and prey protein interactors. The following western blot analysis provides an example of crosslinking regents that are used in vivo.
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.
2. Prepare specific protein conjugates
Heterobifunctional crosslinkers that have an NHS ester group at one end and a different reactive group at the other end (such as a sulfhydryl-reactive maleimide) can be used to create specific protein conjugates.
One example is the conjugation of a purified antibody with horseradish peroxidase enzyme to yield an antibody-HRP conjugate for use in ELISA. Another example is conjugation of peptide antigens to KLH or other carrier protein to prepare an effective immunogen. Sulfo-SMCC is the crosslinker most often use for this method. SM(PEG)n crosslinkers are like SMCC but have different spacer arm lengths based on the number of polyethylene glycol (PEG) units they contain.
Because different functional groups are involved, the reaction can be done in a controlled, step-wise manner. In both of these examples, the amines of one protein can be activated in isolation using the NHS-ester reactive group; when the second protein (bearing sulfhydryl groups) is added, it conjugates to each molecule of the first protein that had been activated. Commonly needed proteins, such as HRP or KLH, are commercially available in pre-activated form (i.e., in which the NHS ester reaction with SMCC is already complete). In this representative experiment, a cysteine coupling assay was used to assess the levels of maleimide activated carrier proteins.
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.
3. Label antibodies and other proteins
The most popular and effective biotinylation and fluorescent labeling reagents for antibodies and proteins are NHS-ester compounds. There are at least two reasons for this popularity, availability and widespread use in labeling applications:
- Biotin and many fluorescent compounds naturally contain or are easily synthesized with a carboxyl group, which can be easily derivatized using carbodiimide chemistry (usually DCC) to produce the NHS-ester compound.
- The most common targets for labeling are large proteins like antibodies (MW of IgG is 150,000), which have 10 to 15 readily available lysine amines with which NHS-ester compounds can react to attach the desired affinity or detection tag.
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.
4. Immobilize antibodies and other proteins
Beaded agarose resin, magnetic particles and several other types of solid supports are available in forms that are activated with NHS-ester groups. These activated supports will stably and efficiently conjugate with proteins or other amine-containing ligands to immobilize them for use in affinity purification procedures. As described above, NHS esters hydrolyze easily; therefore, NHS-agarose and similar activated resins are always supplied dry or slurried in organic solvent (usually acetone).
An important alternative to NHS esters for protein immobilization to agarose beads is aldehyde-activated resin for conjugation by reductive amination. For more information on this related amine-immobilization method (AminoLink Products), see the page on Carbonyl-reactive Crosslinker Chemistry.
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.
Imidoester reaction chemistry
Imidoester crosslinkers react with primary amines to form amidine bonds. Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half life and reactivity with amines increases; therefore, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8-10. Studies using monofunctional alkyl imidates reveal that at pH <10, conjugation can form with just one imidoester functional group. An intermediate N-alkyl imidate forms at the lower pH range and will either crosslink to another amine in the immediate vicinity, resulting in N,N'-amidine derivatives, or it will convert to an amidine bond. At higher pH, the amidine is formed directly without formation of an intermediate or side product. Extraneous crosslinking that occurs below pH 10 sometimes interferes with interpretation of results when thiol-cleavable diimidoesters are used.
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
Homobifunctional imidoester crosslinkers have been used to study protein structure and molecular associations in membranes and to immobilize proteins onto solid-phase supports. The resulting amidine is protonated and therefore has a positive charge at physiologic pH; to some degree, this preserves the native charge properties of the original amines it replaces, and this may be useful in certain experiments. Imidoester crosslinkers also have been examined as a substitute for glutaraldehyde for tissue fixation. Despite their charge properties, imidoesters can penetrate cell membranes and crosslink proteins within the membrane to study membrane composition, structure and protein:protein and protein:lipid interactions. These crosslinkers have also been used to determine or confirm the number and location of subunits within multi-subunit proteins. In these experiments, large molar excesses of crosslinker (100- to 1000-fold) and low concentrations of protein (less than 1mg/mL) are used to favor intramolecular over intermolecular crosslinking.
Although imidoesters are still used in certain procedures, the amidine bonds formed are reversible at high pH. Therefore, the more stable and efficient NHS-ester crosslinkers have steadily replaced them in most applications.
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- Grabarek, Z. and Gergely, J. (1990). Zero-length crosslinking procedure with the use of active esters. Anal Biochem 185:131-5.
- Staros, J.V., et al. (1986). Enhancement by N-hydroxysulfosuccinimide of water-soluble carbodiimide-mediated coupling reactions. Anal Biochem 156:220-2.
- Timkovich, R. (1977). Detection of the stable addition of carbodiimide to proteins. Anal Biochem 79:135-43.
For Research Use Only. Not for use in diagnostic procedures.