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The first cross-linking agents used for modification and conjugation of proteins were bireactive compounds containing the same functional group at both ends.
Glutaraldehyde is the most popular homobifunctional reagents used for protein conjugation, especially for creating antibody-enzyme conjugates.
Amino groups on proteins may react with the bis-aldehyde compound glutaraldehyde to form derivatives able to cross-link with amino-groups on the particle.
When using these homobifunctional reagents for coupling proteins to microspheres, one main disadvantage is the potential for creating cross-links between two proteins leading to undefined complexes and precipitated.
To overcome these problems, two-step reaction was developed, removing the excess of cross-linkers before adding the proteins.
The reaction mechanism for this modification can proceed by different ways. The more simple of these is the formation of a Schiff base linkage between one of the aldehyde ends and amines on proteins (e-amine from lysine or a-amine from most amino acid or primary amine from the particle) to leave the other aldehyde terminal free to conjugate with another molecule.
Schiff base interactions between aldehydes and amines typically are not stable enough to form irreversible linkages, and have to be reduced with suitable reductants.
Glutaraldehyde, used in large excess when compared to the number of amino groups of the particle, is first allowed to react with the NH2 groups at the surface of the bead. (a molar ratio would lead to cross linking of the particles).
The excess of Glutaraldehyde is removed.
The second aldehyde group reacts in a second step with the amino groups of the protein to be bound to the microspheres.
In a Eppendorf tube, add 100 µl of Latex amino-modified microspheres (10% solid)
1°) Add 700 µl of a NaH2PO4 (10 mM) pH 6.8 Add glutaraldehyde to a final concentration of 1.25%
React 4 hrs at room temperature
2°) Wash the activated particles to remove the excess of glutaraldehyde in the same buffer.
Dissolve the protein to be conjugated at a concentration of 1 to 10 mg/ml in a sodium carbonate buffer 0.1 M pH 9.5
1°) Mix the particle suspension with the protein at the desired ratio and react overnight at +4°C
2°) Wash the particles twice in the same buffer
3°) To reduce the Schiff bases and any excess aldehyde, add sodium borohydride to a final
concentration of 10 mg/ml
4°) Reduce for 1 hr at +4°C
5°) Wash the particles twice and resuspend in the desired buffer.
Avrameas, S. (1969) coupling of enzyme to proteins with glutaraledhyde. Immunochemistry 6, 43-52
Avrameas, S. and Ternynck, T. (1969) The cross-linking of proteins with glutaraldehyde and its use for the preparation of immunosorbents. Immunochemistry 6, 53-66
Hermanson, G.T., Mallia, A.K., and Smith, P.K. (1992) in "Immobilised Affinity Ligand Techniques." Academic Press, San Diego
Hermanson, G.T., (1996) in "Bioconjugates Techniques." Academic Press, San Diego
