An alternative method for coupling thiol-containing proteins or antibodies to amine particles is to use a heterobifunctional crosslinker containing an amine-reactive NHS ester at one end and a thiol-reactive maleimide group on the other end. NHS–PEGn–maleimide crosslinkers are available in a number of spacer lengths depending on the size of the polymer chain in the PEG component
Figure.The modification of amine-containing particles with NHS–PEG4–maleimide
1. Wash 10 mg of amine particles into 10-mM sodium phosphate, pH 7.2 (coupling buffer) using centrifugation. Resuspend the particles in 1 ml of the same buffer.
2. Dissolve NHS–PEG6–maleimide (MW 601.6) into DMSO at a concentration of 20-mM. PEG type crosslinkers often exist as a thick oily mass, and preparing the solution may involve dissolving an entire vial of the compound into DMSO to accurately determine the required concentration.
3. Add 50 μl of the NHS–PEG6–maleimide solution to the 1 ml particle suspension and mix thoroughly to dissolve.
4. React for 1 h at room temperature with mixing.
5. Quickly wash the particles with coupling buffer at least twice using centrifugation.
6. 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 on proteins or peptides may be done from disulfides by reduction. Alternatively, a thiolation reagent may be used to add thiols to the protein surface for coupling (see the protocols in Chapter 2, Section 4.1).
7. 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 maleimide reactive sites.
8. Remove excess protein and reactants by washing with coupling buffer at least three times using centrifugation. Storage of the 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