Guidelines for Coupling to Ready-to-Couple™ Chloromethyl microspheres
Chloromethyl microspheres are usually supplied at 10% solids concentration.
Dilute the microspheres to a concentration of 0.5% to 1% solids using a pH 7.0 phosphate buffer.
Note: When diluting to less than 1% solids concentration, it may be necessary to use a pH 7.0 phosphate buffer containing 0.05 to 0.1% surfactant, preferably PolyMicrospheres specialty surfactant for coupling experiments. Make sure that the dispersion is uniform after dilution, and no aggregates are present. Also make sure that the pH of the diluted microspheres don't exceed pH 7.0, otherwise cleavage of the CH2-Cl bond may occur prematurely before the addition of the ligand.
Options: Generally, it is not necessary to clean the microspheres prior to coupling reactions. You may clean the microparticles before coupling if necessary. If you centrifuge the microparticle dispersion for cleaning, decant the supernatant, and redisperse the microparticles using a pH 7.0 phosphate buffer containing 0.05 to 0.1% surfactant, preferably PolyMicrospheres specialty surfactant for coupling experiments. Redispersion may require stirring, shaking, vortexing, and sonication (sonic probe is preferred over sonic bath). Vortexing (30 sec) followed by sonic probe (20 sec) is very effective in redispersing microspheres. Repeat one more time if necessary.
Note: Repeat the washing step one more time if necessary, followed by redispersion. Make sure that the dispersion is uniform after cleaning, and no aggregates are present.
Mix the microparticle dispersion and the ligand solution (pH adjusted to 7.0).
Adjust the pH of the reaction mixture to pH 8.5 by adding 0.1M NaOH solution.
Note: Generally the coupling reaction occurs at pH 8.5±0.5; Depending on the ligand, optimum pH and other conditions for best coupling performance need to be investigated.
Continue the mixing/stirring of the reaction mixture for a minimum of 4 hours; overnight stirring is recommended.
Usual workup: That is wash, and redisperse with the storage buffer. Phosphate buffer at pH 7.0-7.4 could be used as storage buffer. Repeat the washing step one more time, and redisperse with the storage buffer. Redispersion may require stirring, shaking, vortexing, and sonication.
Note: Generally it is not necessary to quench with blocking agents. But sometimes, quenching with blocking agents may be beneficial. Store at 3-4°C.
Proprietary info, PolyMicrospheres