Cross-linking agents contain at least two reactive groups that are reactive towards numerous groups, including sulfhydryls and amines, and create chemical covalent bonds between two or more molecules. Functional groups that can be targeted with cross-linking agents are primary amines, carboxyls, sulfhydryls, carbohydrates and carboxylic acids. Protein molecules have many of these functional groups and therefore proteins and peptides can be readily conjugated using cross-linking agents. Cross-linking agents are used to study protein structure and function, to anchor proteins to solid supports, preparation of immunogens, immunotoxins, and other conjugated protein reagents.
Cross-linking agents can be divided into groups dependent on the number and similarity of the reactive groups:
• Homobifunctional have two reactive ends that are identical.
• Heterobifunctional have two different reactive ends.
Homobifunctional cross-linkers are used in one-step reactions while the heterobifunctional cross-linkers are used in two-step sequential reactions, where the least labile reactive end is reacted first. Homobifunctional cross-linking agents have the tendency to result in self-conjugation, polymerization, and intracellular cross-linking. On the other hand, heterobifunctional agents allow more controlled two step reactions, which minimizes undesirable intramolecular cross reaction and polymerization.
The most widely used heterobifunctional cross-linking agents are used to couple proteins through amine and sulfhydryl groups. The least stable amine reactive NHS-esters couple first and, after removal of uncoupled reagent, the coupling to the sulfhydryl group proceeds. The sulfhydryl reactive groups are generally maleimides, pyridyl disulfides and a-haloacetyls. Other cross-linkers include carbodiimides, which-link between carboxyl groups (-COOH) and primary amines (-NH2). There are heterobifunctional cross-linkers with one photoreactive end. Photoreactive groups are used when no specific groups are available to react with as photoreactive groups react non-specifically upon exposure to UV light.
It is often desirable to minimize the degree of structural shift due to cross-linking reactions, and more so if the protein molecule is biologically active. Therefore, cross-linking is performed under mild buffer and pH conditions. Depending on the application, the degree of conjugation is also important and an optimal cross-linker to protein ratio must be maintained. The number of target groups on the outer surface of a protein is also important. If the exposed target groups are readily available for conjugation; a lower cross-linker to protein ratio can be used.
Cross-linkers are available with different spacer arm lengths. A cross-linker with a longer space arm may be used where two target groups are further apart. The availability of cross-linkers with different spacer arms allows optimization of cross reaction efficiency. Cross-linkers with short space arms are suitable for intramolecular cross-linking. Cleavable cross-linkers are also available which extends the scope of protein analysis.
Selection of Protein Cross-linkers
The following features are taken into consideration when making selection of a cross-linker:
1. Reagent solubility
2. The nature of reactive groups
3. Homobifunctional or heterobifunctional
4. Photoreactive or thermoreactive groups
5. The length of spacer arm
6. Conjugated product cleavable or not
7. Potential for further labeling
8. Reaction condition needed for conjugation
For additional information, such as cross-linking applications, specific cross-linking reaction conditions and other important cross-linking information, download the Protein Crosslinker technical bulletin.
Double Do™ Cross-linkers
G-Biosciences offers a wide selection of cross-linking reagents as Double Do™ Cross-linkers. The Double Do™ Cross-linkers line of products include the chemical cross-linker, supporting accessories consisting of specific reaction buffers, complete reaction systems, selection guides and protocols.
Double Do™ Cross-linker Selection Guide & Ordering Information
To select a cross-linking reagent several factors need to be considered:
• Reactive Toward: Determines the target residues to be cross-linked, select a reagent that does not interfere with protein’s function.
• Membrane Permeability: For cell surface labeling, select non-membrane permeable reagents.
• Cleavable: For easy release of cross-linked proteins from solid supports or for further downstream applications.
• Reversible: An alternative to cleavable reagents are reversible reagents. For example, ANB-NOS is released by photolysis.
• Steric Hinderance: Bulky groups around the binding site may require reagents with longer spacer arms.