Bioconjugation

What is Bioconjugation?

Bioconjugation is the chemical linking of two biomolecules to form a single hybrid that retains the biological activity of each component, yet provides a novel function that is not possible with each individual biomolecule. Most complex biomolecules such as proteins exist and function only in aqueous environments. For this reason, the preparation of bioconjugates must be carried out in aqueous solutions, and any suitable bioconjugation chemistry must preserve the biological activity and function of the biomolecules in this type of environment.

Bioconjugation workflows differ depending on the intended application, capturing a biomolecule or conjugating a biomolecule. Vector Laboratories has products to assist you with both.

How is bioconjugation performed?

Conjugates are generally formed through the addition of separate but complementary functional groups to each of the two biomolecules. These functional groups are typically introduced through a process called modification, which consists of attaching linkers to an amine or thiol group present on the biomolecules of interest. The two modified biomolecules are then mixed together to form the desired bioconjugate via the complementary linkers incorporated during modification.

What are some considerations when performing bioconjugation?

When choosing a conjugation strategy, it is important to consider the characteristics that make a desirable bioconjugation technology. The following list presents the criteria required for development:

  1. Linkers must be incorporated on biomolecules in a mild, controllable reaction in aqueous solution, in a manner which maintains the biomolecules’ inherent function.
  2. Conjugate bonds should only form between the two complementary linkers incorporated on biomolecules during modification, and not through endogenous functional groups found within the biomolecules themselves.
  3. Linker incorporation and conjugate formation should be easily quantifiable through simple and non-destructive methods such as spectrophotometry.
  4. Linker-modified biomolecules and the conjugate linkage itself should be stable under a broad pH range and at elevated temperatures.
  5. Conjugation reaction kinetics should be fast and stoichiometrically efficient.
  6. Linkers should be easily incorporated on a variety of biomolecules, including oligos and peptides during solid phase synthesis.
  7. The conjugation reaction should occur directly upon mixing the two modified biomolecules, without the addition of any agent that could disrupt their function such as an oxidant, reductant, or metal.
  8. There should be no undesirable covalent side reactions during modification or conjugation.
SoluLINK<sup>®</sup> Bioconjugation Technology
 

What is SoluLINK bioconjugation technology?

SoluLINK bioconjugation technology mildly, efficiently, and reproducibly conjugates and immobilizes all categories of biomolecules, including proteins, peptides, oligonucleotides, carbohydrates, drugs, and surfaces. This chemistry is based on the reaction of an aromatic hydrazine with an aromatic aldehyde, which forms a stable bis-arylhydrazone conjugate bond. When functionalizing amines, S-HyNic is used to incorporate 6-hydrazinonicotinamide (HyNic) groups. This linker possesses a succinimidyl ester (NHS ester) that readily reacts with amino groups on proteins, other amine-containing biomolecules, and surfaces using standard NHS ester reaction conditions. The aromatic aldehyde is incorporated on biomolecules with S-4FB in the same manner.

What are the advantages of using SoluLINK bioconjugation technology?

One of the key advantages in using the SoluLINK bioconjugation technology is the incorporation of the bis-arylhydrazone bond and its ability to form a traceable chromophore. This chromophore absorbs maximally at 354 nm with a molar extinction coefficient of 29,000 L·mol-1·cm-1 and has been demonstrated to be both heat and pH stable (95°C for 2 hours; pH 2–10). These features allow for:

  1. Facile quantification of the number of linkers incorporated on each biomolecule prior to conjugation.
  2. Monitoring of the conjugation reaction in real time to assess its progress.
  3. Direct quantification of the number of linkages formed in the conjugate and the precise number of biomolecules or ligands attached.
  4. Visualization of the conjugate peak during FPLC or HPLC purification and identification of fractions containing the desired conjugate.
  5. Easy and non-destructive quantification.

Further advantages over azide-alkyne “click” chemistry or maleimide-thiol coupling include:

  1. No requirement for heavy metal catalysts.
  2. Faster conjugations that increase labeling efficiency and lower reagent costs.
  3. Superior stability in aqueous phases.
  4. No need for the use of reducing agents
  5. Eliminates the concern for homo-dimer formation such as disulfide bridge formation.
 

Additional resources available from Vector Laboratories

Bioconjugation White Paper

Bioconjugation White Paper from Vector Laboratories

IHC Multiplexing Guide

IHC Multiplexing Guide

Enzyme Substrates

Enzyme Substrates poster