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Protein Crosslinking Applications

Crosslinking is the process of chemically joining two or more molecules by a covalent bond. Crosslinking reagents (or crosslinkers) are molecules that contain two or more reactive ends capable of chemically attaching to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules.

Crosslinking reagents are used in a variety of techniques to assist in determining partners and domains of protein interactions, three-dimensional structures of proteins, and molecular associations in cell membranes. They are also used to immobilize proteins on solid supports for affinity purification, to conjugate haptens to carrier proteins for immunization, and to prepare antibody-enzyme conjugates for detection procedures. This article briefly describes some of these crosslinking applications.

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Protein Methods Library Home

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Crosslinking Technical Handbook

Protein Structural and Subunit Analysis

Crosslinkers can be used to study the structure and composition of proteins in purified or complex samples. Experiments using different amine-, carboxyl- or sulfhydryl-reactive reagents can assist in identification and quantification of particular amino acids or for determination of the number, location and size of subunits.

Experiments with different lengths of otherwise identical crosslinkers can reveal the molecular distance between particular functional groups in the secondary, tertiary or quaternary protein structure. For example, if a very short amine-to-amine crosslinker does not successfully crosslink subunits of a dimeric protein but a slightly longer crosslinker does, one can conclude that the relevant amino acids in the native dimeric structure exist at a distance approximately equal to the length of the longer crosslinker.

Non-cleavable, homobifunctional, sulfhydryl-reactive linkers such as BMH can be used to convert cleavable disulfide bonds between subunits into permanently (nonreducible) crosslinks. In some circumstances, the crosslinking pattern or success may be affected by the crosslinker’s solubility and accessibility to the microenvironment of a protein's molecular structure. For example, hydrophobic crosslinking reagents tend to react more effectively in hydrophobic regions of molecules.

Assessment of crosslinking results is usually accomplished by some form of electrophoresis (1D or 2D), followed by staining or Western blot detection. Analysis by both non-reducing and reducing polyacrylamide gel electropheresis (PAGE) is informative when crosslinkers containing reducible disulfide bonds in their spacer arms were used. Alternatively, using matched cleavable and noncleavable pairs of crosslinkers can also provide this information.

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Crosslinker Application Guide

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Crosslinkers at a Glance
(Table of all crosslinkers)

Crosslinker Selection Guide
(Interactive guide)

Protein Interaction Analysis

Protein-protein interactions are the basis for nearly all cellular pathways, and the discovery and characterization of protein interactions is increasingly important in proteomics research. Because most protein interactions are transient (and often labile) events, crosslinking techniques are important tools for capturing and stabilizing them so that they can be analyzed.

Some of the same strategies for crosslinking and analysis that were described above for subunit analysis can be used for protein-protein interaction analysis. These include testing cleavable and noncleavable pairs of different length homobifunctional crosslinkers and then assessing the results by gel electrophoresis. If a specific antibody or other probe is available for at least one protein component of the interaction, then the covalently bound complex of proteins can be co-purified and/or detected in Western blots as a means of evaluating the results.

Protein interaction experiments can be performed with purified proteins, but it is more common to investigate and attempt to characterize the interactions in vivo using cells grown in specific treatment conditions. Successful crosslinking corresponding to different time-points after treatment indicates when a given interaction occurred in the cellular pathway responsible for the response. A number reagent options for in vivo crosslinking are available, including hydrophilic and hydrophobic varieties to concentrate reaction at the cell surface or within cell membranes, respectively.

Heterobifunctional crosslinkers, especially those having one end that is non-specific and photo-activatable, are particularly useful for protein interaction analysis. These linkers can be reacted first to a purified "bait" protein 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.

When the heterobifunctional, photo-reactive crosslinker also contains a detectable tag (e.g., biotin, a radioactive isotope or mass variant) and a cleavable spacer arm, the reagent can be used to transfer the detectable tag from known bait protein to unknown prey protein. This is called label transfer. See related articles about Protein Interactions Analysis for more information about label transfer and other crosslinking method for studying protein interactions.

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Overview of Crosslinking

Chemistry of Crosslinking

In vivo Crosslinking

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Label Transfer Reagents

NHS-Diazirine Crosslinkers

Photo-reactive Amino Acids

Immunotoxin Construction

Crosslinkers provide a means to conjugate tumor-specific antibodies to toxic molecules that can then used to target antigens on cells. These "immunotoxins" are brought into the cell by surface antigens and, once internalized, they proceed to kill the cell by ribosome inactivation or other means. The type of crosslinker used to make an immunotoxin can affect its ability to locate and kill the appropriate cells. For immunotoxins to be effective, the conjugate must be stable in vivo. In addition, once the immunotoxin reaches its target, the antibody must be separable from the toxin to allow the toxin to kill the cell. Cleavable disulfide-containing conjugates have been shown to be more cytotoxic to tumor cells than noncleavable conjugates of ricin A immunotoxins. Cells are able to break the disulfide bond in the crosslinker, allowing the release of the toxin within the targeted cell.

SPDP is a reversible NHS-ester, pyridyl disulfide crosslinker used to conjugate amine-containing molecules to sulfhydryls. For several years, this has been the “workhorse” crosslinker for production of immunotoxins. The amine-reactive NHS-ester is usually reacted with the antibody first. In general, toxins do not contain surface sulfhydryls; therefore, sulfhydryls must be introduced onto them by reduction of disulfides, which is common for procedures involving ricin A chain and abrin A chain, or through chemical modification reagents. A second SPDP molecule can be used for this purpose and is reacted with amines on the immunotoxin, then reduced to yield sulfhydryls. Another chemical modification reagent that is commonly used for production of immunotoxins is 2-iminothiolane, also known as Traut’s Reagent, which reacts with primary amines to add sulfhydryl groups.

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Bioconjugate Techniques, Greg Hermanson
Bioconjugate Techniques,
2nd Edition (2008)
Greg T. Hermanson,
Academic Press, Inc.,
1202 pages.

Immunogen Preparation

Specific antibodies are created by injecting animals with peptide or other antigens and then harvesting the antibodies produced by the animal in response to the foreign molecule. Peptides and other antigen molecules are not sufficiently complex or large to evoke the necessary immune response that is responsible for producing antibodies. Therefore, these small antigens (called haptens) must be attached to larger proteins or molecules to create an effective immunogen.

Many crosslinkers can be used to conjugate haptens and carrier proteins to create immunogens. The best crosslinker to use depends on the functional groups present on the hapten and the ability of the hapten-carrier conjugate to function successfully as an immunogen after its injection. The carbodiimide EDC is effective for producing peptide-carrier protein conjugates because both proteins and peptides contain several carboxyls and primary amines.

When peptide antigens are synthesized with terminal cysteines, they can be attached to supports or to carrier proteins using sulfhydryl/amine-reactive, heterobifunctional crosslinkers (e.g., Sulfo-SMCC). This method is provides for efficient and controlled two-step conjugation while avoiding internal crosslinks to the peptide antigen that block or alter its presentation as an antigen.

Certain drug compounds and other unusual haptens require crosslinking methods with specialized reactivities. For most situations, however, immunogen preparation is easy to perform using pre-activated immunogenic carrier proteins (KLH, BSA, etc.) and conjugation kits based on EDC or Sulfo-SMCC crosslinker chemistries.

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Antibody Production

 

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Immunogenic Carrier Proteins for Antibody Production

Protein-Protein Conjugation

One of the most-used applications for crosslinkers is the production of protein-protein conjugates. Indeed, most secondary antibodies used in ELISA, Western blotting and other kinds of immunodetection methods are examples of protein-protein conjugates. Antibody-enzyme conjugates are prepared by crosslinking purified antibody and enzyme proteins. Horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most popular and versatile reporter enzymes for making secondary antibody probes. The same two enzymes are also commonly used to make conjugates with streptavidin and other biotin-binding proteins.

Because secondary antibody- and streptavidin-enzyme conjugates are commercially available in nearly every conceivable variety, few situations exist in which these particular conjugates would need to be prepared by individual researchers. However, primary antibodies and specialized proteins of research interest may not be available commercially. The same methods of crosslinking that are used by manufacturers of conjugated secondary antibodies can be applied at a smaller scale by individual researchers for many kinds of protein-protein conjugation needs.

Glutaraldehyde is an aggressive, indiscriminant crosslinking reagent that was commonly used in the past to prepare antibody-enzyme conjugates. When used in large molar excess, glutaraldehyde can be used to activate one protein (e.g., HRP) for conjugation to the second protein (e.g., the antibody). Conjugates are easy to make with this method but often partially inactivate the enzyme or antibody and produce high background in immunoassays.

Homobifunctional NHS-ester or imidoester (amine-reactive) crosslinkers can be used in a one-step procedures to conjugate two purified proteins, but there is no way to prevent polymerization and self-conjugation, which are as likely to occur as the desired intermolecular conjugation. Keep in mind that, unlike in protein interaction applications, the two proteins being conjugated in the case of an antibody and enzyme do not associate together in solution; two antibody molecules or two enzyme molecules are as likely to encounter each other as an antibody and enzyme molecule.

If one protein is a glycoprotein and its sugar groups are not essential to its function (as in the case of HRP), then the carbohydrate moieties can be used as the basis for conjugation. Oxidation of carbohydrate groups with sodium meta-periodate yields aldehyde groups that are directly reactive toward primary amines (i.e., side chain of lysine residues) via reductive amination. These conjugates often result in less background in enzyme immunoassays and are relatively easy to prepare; however, some self-conjugation of the antibody may occur.

Heterobifunctional crosslinkers are perhaps the best choices for antibody-enzyme or other protein-to-protein crosslinking. Unwanted self-conjugation inherent when using homobifunctional NHS-ester reagents or glutaraldehyde can be avoided by using a reagent such as SMCC or Sulfo-SMCC. In separate reactions, one protein can be reacted to the amine-specific end of this reagent while the other protein is treated with reducing agent or sulfhydryl-addition reagent to expose or created sulfhydryl groups. Finally, after removing excess non-reacted reagents, the two proteins can be mixed to allow the sulfhydryl-reactive (maleimide) groups of the first protein to conjugate with the sulfhydryl groups of the second protein.

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Crosslinker Application Guide

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Sodium meta-Periodate

HRP Conjugation Kits:
Maleimide-Activated HRP Kit
Periodate-Activated HRP Kit
Glutaraldhyde-Activated HRP Kit


Amine-to-Sulfhydryl Linkers:
SMCC
Sulfo-SMCC
Browse Similar Reagents


Sulfhydryl-Addition Reagents:
Traut's Reagent (2-Iminothiolane)
SATA and SATP
Sulfo-LC-SPDP
 

Solid-Phase Immobilization

Proteins, peptides and other molecules can be immobilized onto solid supports for affinity purification of proteins or for sample analysis. The supports may be nitrocellulose or other membrane materials, polystyrene plates or particles, beaded agarose or polyacrylamide resins, or glass slides. Some supports can be activated for direct coupling to a ligand.

We offer a variety of affinity resin supports and several kinds of polystyrene plates and glass slides that are pre-activated or functionalized with chemical groups to allow direct or crosslinker-mediated immobilization of proteins and other ligands. For example, Thermo Scientific SulfoLink Coupling Resin is beaded agarose that contains sulfhydryl-reactive iodoacetyl groups; cysteine-containing peptides are easily immobilized for use in affinity purification methods. Another example is Thermo Scientific CarboxyLink Coupling Resin whose agarose backbone has been derivatized with diaminodipropylamine (DADPA) to present numerous primary amines; carboxyl-containing peptides are easily crosslinked to the amino using EDC.

Immobilization techniques are discussed in greater detail in other articles in the Protein Methods Library. In general, heterobifunctional chemistries involving amine-to-carboxyl or amine-to-sulfhydryl strategies are most successful and versatile. By combining particular resins or functionalized surface materials with different kinds of crosslinkers and modification reagents, one can accomplish all sorts of specialized immobilization scenarios.

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Covalent Immobilization Methods for Affinity Ligands

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Tech Tip #5: Attach an antibody onto glass, silica or quartz surface

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Activated Agarose Supports
for Protein Immobilization


Amine-binding Microplates
Sulfhydryl-binding Microplates

DNA/RNA Crosslinking to Proteins

Crosslinking of DNA or RNA to proteins is more limited because the reactivities of most crosslinkers favor protein-protein crosslinking over protein-DNA crosslinking. To assist in these crosslinking methods, DNA probes are often synthesized with primary amines or thiols attached to specific bases. After insertion of the bases into DNA, amine- or sulfhydryl-reactive crosslinkers can be used for their conjugation to proteins.

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Overview of Protein-Nucleic Acid Interactions

ChIP Assay Procedure and Essential Tools

Other Applications

There are many additional applications for crosslinkers that are either antiquated methods, new technologies or for more specialized needs. Older methods for peptide synthesis involve use of carbodiimide crosslinkers such as DCC and EDC for the step-wise addition of individual amino acids to support bound peptides. Crosslinkers such as glutaraldehyde and dimethylpimelimidate have been used for tissue fixation.

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Crosslinkers at a Glance
(Table of all crosslinkers)

Crosslinker Selection Guide
(Interactive guide)
Written and/or reviewed by Douglas Hayworth, Ph.D.

Instructions | MSDS | CofA
Product Instructions | MSDS | CofA  

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