SpheroTechnical Notes #16 - Covalent Coupling of Proteins to Microbeads Using a Heterobifuntional Coupling Agent

Covalent coupling of lysozyme to amino-polystyrene particles utilizing a heterobifunctional coupling agent, SPDP, and to aldehyde particles was studied using Particle Concentration Fluorescence Immunoassay (PCFIA). The results showed that these methods can be used to couple lysozyme covalently to amino-polystyrene and aldehyde particles.

The modification of amino-polystyrene particles with SPDP offers the advantage that the resulting pyridyldisulfide particles can be used to react specifically with the sulfhydryl group of proteins, or they can be reduced further with DTT to form sulfhydryl particles. The aldehyde particles can be used to couple covalently to proteins without any coupling agent.

The activity of the lysozyme coated particles depends upon the orientation of lysozyme on the particles. Higher activity was obtained when the amino groups of the lysozyme were utilized to couple to the particles covalently.
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SpheroTechnical Note #16 will provide the following information:
  • A protocol for covalently coupling lysozymes to amino-polystyrene particles through either EDC coupling on SPDP modification
  • A protocol for the covalent coupling of lysozymes to aldehyde particles without the use of a coupling agent
  • How the activity of coated particles varies with the coupling methods used and depends on the orientation of lysozyme on the surface of the microparticles.
  • Various coupling strategies including passive adsorption on particles with different surface charges and covalent coupling using different functionalized particles and coupling agents can be used to optimize the activity of immobilized protein on microparticles.