2) The reaction is efficient: The reaction is very stoichiometrically efficient as input of only 1-2 moles of second protein/mole first protein is required for complete conversion to conjugate.
3) The conjugate bond is extremely stable: the bis-arylhydrazone conjugate bond is stable up to 92℃ and pH 2.0-10.0.
4) The reaction conditions are extremely mild and do not cause antibody denaturation: unlike thiol-based conjugation protocols where reducing reagents are required that can compromise the activity of proteins by cleaving disulfide bonds, the HyNic-4FB conjugation couple leaves disulfide bonds intact. No metals, oxidizing, or reducing reagents are required.
5) The conjugation is traceable spectrophotometrically: the HyNic-4FB conjugate bond is chromophoric; it absorbs light at 354 nm and has a molar extinction coefficient of 29,000 L/(mol*cm).
6) The modifications of both the HyNic moiety on the protein and the 4FB moiety on the protein is quantifiable using a colorimetric assay: the reproducibility of any reaction is dependent on accurate characterization of all components. The Molar Substitution Ratio (MSR; i.e. the number of HyNic groups incorporated per protein) can be quantified colorimetrically as a reaction with 2-sulfobenzaldehyde yields a chromophoric product that absorbs at 350 nm with a molar extinction coefficient of 28,500 L/(mol*cm). The MSR of 4FB groups can be determined colorimetrically by its reaction with 2-hydrazinopyridine forming a hydrazone that absorbs at 348 nm with a molar extinction coefficient of 24,500 L/(mol*cm). This kit contains all the reagents necessary to determine both MSRs. Procedures to guide users through this process are given in the protocol below.
The Keys to Successful Conjugation
The following are three crucial requirements that must be fulfilled for a reproducibly successful preparation of a protein-protein conjugate using the SoluLINK bioconjugation technology:
1. Desalting: prior to modification, the starting proteins must be thoroughly desalted, removing all amine contaminants and exchanging the proteins into 1X Modification Buffer.
2. Protein concentration: the recommended protein concentrations must be adhered to in all steps.
3. Molar substitution ratio: the molar ratio of HyNic on the protein and 4FB on the protein must be determined and within the desired range before continuing to the next step.