Carboxyl Polystyrene Particles with avidin or other proteins using EDC covalent coupling

Coupling Principle

Carboxylated polystyrene particles conjugated with proteins and antibodies have low reaction costs, short reaction times, and require specific chemical reagents (EDC and NHS) for activation. If the stability of the activation intermediate is poor, it will affect the coupling efficiency, and the residual COOH will cause non-specific adsorption.

EDC is a zero-length crosslinking agent for the reaction between carboxyl and amino groups, which plays a role in activating carboxyl groups. EDC reacts with carboxyl groups to form an amino-activated O-acyl isourea intermediate, which can rapidly react with amino groups to form amide bonds and release isourea by-products. This medium is unstable in water. Therefore, the two-step co-reaction relies on N-hydroxysuccinimide (NHS) to stabilize the structure. Meanwhile, NHS has a strong absorption peak at 280 nm and can be used for the determination of protein coupling rate. The failed reaction with the amino group leads to the hydrolysis of the medium, the regeneration of the carboxyl group, the release of N-generation urea, and the side reaction forms N-acylion, which usually limits the hydrophobic region of the carboxyl localization protein.

Coupling Step

Carboxyl group activation on the surface of carboxylated polystyrene particles

After thoroughly mixing the carboxylated polystyrene particles, take an appropriate amount of the carboxylated polystyrene particles into a centrifuge tube, remove the supernatant, and wash the carboxylated polystyrene particles three times with the prepared 20 mMMES buffer (20 mMMES buffer, pH 5.0, 0.05% Tween 20). Take an appropriate amount of freshly prepared EDC solution (or add an equal volume of NHS solution) to disperse carboxylated polystyrene particles, and activate them at 25¡ãC for 3 h.

Carboxylated polystyrene particles are coupled with biomolecules

Discard the supernatant, disperse an appropriate amount of biomolecules in a buffer solution with pH 8.0, and re-disperse the carboxylated polystyrene particles with this solution. Couple at 25¡ãC for 3 h or overnight at 4¡ãC. After coupling, remove the supernatant, add a buffer containing 1%BSA, re-disperse the carboxyl-polystyrene particles, and couple at 25¡ãC for 1 h to seal the unreacted activated carboxyl groups.

After sealing, the supernatant was removed. The carboxylated polystyrene particles were washed three times with 20 mM pH 7.4 PBS buffer and re-dispersed. They were stored at 4¡ãC. If necessary, 0.02% sodium azide could be added to inhibit bacterial growth.

Coupling Principle

The density of carboxylated polystyrene microspheres is relatively high, and they tend to sink when left standing for a long time. Therefore, please shake them thoroughly before use to obtain a uniform microsphere suspension.

Storage

Storage conditions: Store at 2 to 8¡ãC. Do not freeze or dry.