Figure. The crosslinker SPDP can be reacted with amine particles to create thiol-reactive pyridyl disulfide groups on the surface. Thiol-containing proteins or other thiol molecules can be reacted with these activated particles to result in disulfide linkages, which are reversible by reduction.
1. Wash 10 mg of amine particles into 10-mM sodium phosphate, pH 7.2, using centrifugation. Resuspend the particles in 1 ml of the same buffer.
2. Dissolve SPDP in dimethylformamide (DMF) at a concentration of 6.2 mg/ml (makes a 20-mM stock solution). Add 50 μl of the SPDP solution to the 1-ml particle suspension and mix to dissolve. Note: The small quantity of DMF in a polymeric particle suspension should not affect particle stability, even if the polymer type is susceptible to swelling in pure DMF. Other particle types, such as metallic or silica based, usually are not affected by organic solvent addition, unless their surfaces are non-covalently coated with a dissolvable polymer.
3. React for 30 min at room temperature with mixing.
4. Wash particles with coupling buffer at least three times using centrifugation and resuspend in the same buffer using a sonic probe to fully disperse the particles.
5. Add 1 to 10 mg of a protein or antibody containing an available thiol group to the particle suspension. Alternatively, add the protein to be coupled to the particle suspension in an amount equal to 1- to 10-times molar excess over the calculated monolayer for the protein type to be coupled. The optimal amount of protein to be added should be determined experimentally. Creating thiol groups from disulfides in proteins may be achieved by using a reducing agent or through the use of a thiolation reagent (Chapter 2, Section 4.1).
6. React with mixing for 2 h at room temperature. At the completion of the reaction, cysteine may be added at 50-mM to block excess pyridyl disulfide reactive sites.
7. Remove excess protein and reactants by washing with coupling buffer at least three times. Storage of particles may be done in a suitable buffer at 4°C containing a preservative.
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Reference: Bioconjugate Techniques, 3rd Edition - July 25, 2013, Greg T. Hermanson