Troubleshooting Guides

Bioconjugation technology facilitates the covalent linking of biomolecules, such as proteins, carbohydrates, and nucleic acids, with exogenous fractions, proving invaluable for targeted biotherapeutics, high-throughput drug screening, ligand discovery, disease diagnosis, and biosensors. Despite its potential, the development of new bioconjugates presents several challenges stemming from the complexity of bioconjugation chemistry. This guide outlines some common issues of bioconjugation alongside possible solutions. For additional support, please do not hesitate to contact us.

Fig.1 (Creative Biolabs Original)

Problems Associated with Protein Conjugation

Issue	Possible Cause	Recommended Solution
Lack of site specificity	Multiple reaction sites exist, such as lysine-based conjugation.	Use a catalyst to promote site-specific conjugation of proteins, such as chemoenzymatic labeling in proteins. Incorporate unnatural amino acids into the protein structure, like specific incorporation of unnatural amino acids at the N-terminus.
Unable to access reactive sites	In natural proteins, the structure and folding of proteins may result in the desired reaction groups being limited or unable to react.	Search for alternative reactive sites. Directly modify proteins within acceptable limits to obtain usable reactive sites for the target.

Problems Associated with Protein-DNA Conjugation

Issue	Possible Cause	Recommended Solution
Low yields of conjugates	The reaction conditions are inappropriate.	Adjust your reaction conditions, such as temperature, time, and solvent, to see if this improves the yields. If the problem persists, consider searching for similar reactions in the literature and adjust accordingly.
Low yields of conjugates	The conjugation products are lost in the purification step.	Try another viable purification technique such as biotin displacement assay.
Introduction of reactive sites on DNA	Lack of reactive sites on DNA.	Add desired functional groups to the structure of DNA by direct chemical reaction. Modify the DNA chains by chemical synthesis to add the desired functionality to the structure. For example, phosphoramidite chemistry allows the introduction of functional handles into DNA, which is the most common method for DNA oligonucleotide synthesis.

Problems Associated with Antibody Conjugation

Issue	Possible Cause	Recommended Solution
Low yields of conjugates	Impurities contained in low-purity antibodies may compete with the target antibody for labeling, thus interfering with the conjugation reaction.	Try to use antibodies with >95% purity for the conjugation reaction.
	Irritating reagents usually cause antibody degradation.	Look for milder alternative reagents that can be used to create the same chemical linkage between the target antibody and the molecule. Adjust the reaction conditions by trying to perform the reaction in a colder environment to control the reaction temperature, or in an inert atmosphere (e.g., nitrogen).
	Antibody buffers or additives are not compatible with crosslinkers.	Please verify the antibody formulation utilized for conjugation, as common buffer additives (e.g., Tris, glycine, and azide) may interfere with the conjugation reaction. For example, N-hydroxysuccinimide (NHS) esters are incompatible with primary amine-containing buffers like Tris and glycine due to competitive reactions. If these conditions are present in your antibody, buffer exchange is recommended to remove low molecular weight additives. Routine methods for buffer exchange include dialysis, ultrafiltration, and gel filtration chromatography.
Poor stability of conjugates	The storage condition is incorrect.	Most bioconjugates should be stored at -20 ~ -80°C to minimize degradation. However, freezing may adversely affect certain antibody conjugates. For example, those antibody conjugates containing phycoerythrin (PE) are best stored at 2-6°C. Please store your bioconjugation compounds in strict accordance with the product instructions or references. Additionally, to prevent damage from repeated freezing and thawing, it is advisable to store antibody conjugates in aliquots.
Poor stability of conjugates	The inherent instability of antibodies.	Add a suitable stabilizer to your antibody bioconjugates to extend their shelf life.

Problems Associated with Antibody-Enzyme Conjugation

Issue	Possible Cause	Recommended Solution
Low enzyme activity of conjugates	Linker length affects the stability and reactivity of conjugates.	Try conjugation experiments using crosslinkers with different lengths. Look for alternative crosslinkers that are stable over time. Avoid crosslinkers that break easily under reaction conditions. Avoid enzymatically cleavable bonds, such as peptide-based crosslinkers.
Low enzyme activity of conjugates	The reagents cause degradation of the active enzyme.	Use alternative reagents for your experiments. Use enzyme stabilizers to minimize the degradation of the bioconjugates. Change reaction conditions, such as solvent and pH. Search the literature for alternative conjugation strategies.

For research use only. Not intended for any clinical use.