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Fina Biosolutions offers another Ready-to-Conjugate^TM carrier protein, CRM₁₉₇-Azide to facilitate conjugation by click chemistry.

CRM₁₉₇-Azide, prepared from Finabio’s CRM₁₉₇ (EcoCRM®), is derivatized with approximately 15 azide groups ready for conjugation to alkyne, DBCO, or BCN-tagged ligands including carbohydrates, peptides, proteins, and other molecules of interest. Ready-to-ConjugateTM CRM₁₉₇-Azide has been purified to remove any trace of azide labeling reagent. The product is provided at 5 mg/ml in PBS pH 6.8 containing 5% sucrose at -80°C, and conjugation can be performed without further buffer exchange.

As an example of the utility of CRM₁₉₇-Azide, copper-free click chemistry was reacted DBCO-activated Alexa Fluor 488 and 555 at 2-10x molar ratios in PBS, pH 7.4 (Figure 1A). Following purification, the molar ratio of Dye/CRM₁₉₇ in the conjugates was determined (Figure 1B). The figure clearly shows that DBCO-dyes coupled with CRM₁₉₇-azide with good yield, about 65% for DBCO-AF488 and about 40% for DBCO-AF555. The number of dyes conjugated to CRM₁₉₇ increased linearly with the number of DBCO-dyes used in the conjugation, indicating the azide groups on CRM₁₉₇ were not saturated.

Figure 1. DBCO-AF488 and DBCO-AF555 were conjugated to CRM₁₉₇-azide at molar ratio of Dye/CRM₁₉₇ of 2-10x molar ratios. 25 μl of DBCO-dye/DMSO solutions of desired concentrations to a mixture of 200 μl (1 mg) of CRM₁₉₇-azide and 175 μl of PBS pH 7.2. The reactions were mixed at 20°C for 2 – 4 hours, then incubated at 37°C overnight and purified by dialysis. The Dye/ CRM ratios were calculated from the absorbances.

The ligand density in the conjugates can be controlled by adjusting the DBCO-ligand/CRM₁₉₇ ratios in the conjugation reaction. It is possible to attach as high as 15 ligand molecules to CRM₁₉₇ by performing conjugation with over-saturating DBCO-ligands, depending on the nature of ligands and the coupling conditions. It is recommended that the optimal ligand density determined first at a small scale for the intended application.