1) Weigh out 100 mg of silica beads.
2) Wash the silica beads 3 times with EtOH; pellet the bead on a centrifuge for 2 min at 750 x g, remove the supernatant. Bring the beads up in EtOH and repeat.
3) Make a 50 mM solution of Hynic silane (10 mg) in EtOH (500 µL), add 2% (10 µL) water to the solution to dissolve any remaining solids. This may require intensive vortexing to get the silane into solution.
4) Add the Hynic silane solution to the washed bead pellet such that the silane/silica ratio is 20% w/v (500 µL).
5) Vortex the bead sample and incubate at room temperature on a rotator for 30 min.
Note: Check the pH periodically with pH paper and be sure that it doesn’t go below pH 7.4 during the incubation steps!! Bring the pH to above 7.4 with 1 M NaOH if the pH drops.
6) Add an additional 2% (10 µL) water to the bead solution and continue the incubation for 15 min.
7) Add an additional 10%(50 µL) water to the bead solution and continue the incubation for 5 min.
8) The washing step is very important: Washed the beads 3X each with water, ethanol, water, PBS and Conjugation Buffer (100mM sodium phosphate, 150 mM NaCl, pH 6.0) in that order, using the spin protocol from step 1.
9) Check the supernatant to see if there is a significant A280 from the Hynic-silane; add 100 µL of supernatant to 900 µL of Buffer. The A280 should give a reading no higher than 0.05.
10) Bring the beads up in Conjugation Buffer such that the solution is a 20% w/v beads in Conjugation Buffer.
The Hynic-modified beads are now ready to be conjugated the 4FB-modified biomolecule. For large biomolecules (proteins and antibodies), 20 µg of protein/mg of bead is recommended for maximum conjugation. Allow the beads to conjugate with the protein overnight at room temperature on a rotor or shaker.