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CROSSLINKERS

Introduction

CROSSLINKING REAGENTS are chemical compounds used to covalently bind, or conjugate, biomolecules together. Cyanagen’s crosslinker reagents are classified according to the reactive groups, their hydrophobicity or hydrophilicity, the length of the spacer between the reactive groups. They can be homobifunctional or heterobifunctional, membrane permeable or impermeable. The spacer arm can also be cleavable.

Longer spacer arms are usually more effective for coupling larger proteins or those where the reactive side-chains are sterically protected.

Features

  • Different spacer arms
  • High purity

Crosslinking reagents are chemical compounds containing at least two reactive groups that are reactive towards numerous groups, creating chemical covalent bonds between two or more molecules. This strategy includes the conjugation of proteins, antibodies, nucleic acids and other biomolecules such as receptor binding proteins, hormones or peptides with each other or with any molecular group that adds useful properties such as drugs, toxins, fluorophores, photoprobes, inhibitors and enzymes. In addition to the two reactive groups a wide variety of different types of connectors (or spacer arms) have been developed.
Longer spacer arms are usually more effective for coupling larger proteins or those where the reactive side-chains are sterically protected.

The conformational flexibility and hydrophilicity of the spacer arm is also an important consideration. Homobifunctional crosslinkers (two reactive ends are identical) are used in one-step reactions while the heterobifunctional crosslinkers (two different reactive ends) 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. Heterobifunctional agents allow more controlled two-step reactions, which minimize undesirable intramolecular cross-reaction and polymerization.

When making the selection of a crosslinking agent is suggested to take in consideration the following properties:

  • Reactive toward: determines the target residues to be cross-linked, select a reagent that does not interfere with protein’s function
  • Cleavable by: for easy release of cross-linked proteins from solid supports or for further downstream applications
  • Membrane permeability: for cell surface labeling select non-membrane permeable reagents
  • The length of spacer arm: aliphatic chains tend to fold on themselves in an aqueous environment. Spacers containing structures that are more rigid can be used instead. However, these structures tend to be very hydrophobic. Attachment of a spacer containing an alkylether chain, such as polyethylene oxide (PEO), can endow proteins with many desirable attributes like enhanced aqueous solubility, reduced immunogenicity, enhanced proteolytic resistance, reduced toxicity, improved thermal and mechanical stability.