1°) Washing twice MS at 10 % with wash or coupling buffer: pH 8-9 ( 1 ml in 10 ml). Buffer : avoid amine-containing buffers such as Tris or Glycine, which will compete with the ligand for coupling sites.

2°) Resuspend in 9.5 ml of activating buffer. Buffer : Sodium Carbonate 2M (vortex, ultrasound or rolling).

3°) Dissolve ( in a fume hood) 1.0 g of CNBr (for 100 mg of MS) in 0.5 ml acetonotrile.

4°) Add CNBr solution to the stiring MS suspension allowing the activation reaction for precisely 2 minutes at room temperature ; Concentration of MS suspension is now 10 mg/ml.

5°) Wash quickly the activated MS in a large volume of ice-cold water, then with cold coupling buffer. Resuspend MS in 5 ml of coupling buffer (4°C). Dissolve the ligand to be coupled in 5 ml of coupling buffer at a concentration corresponding to a 1-10 X excess of calculated monolayer.
Combine MS suspension and protein solution.

6°) Keep suspension at 4°C for 24 hours with constant mixing.

7°) Wash, resuspend in 10 ml of quenching solution, and mix gently for 30 minutes. Quenching solution with primary amine source: 30-40 mM (hydroxylamine, ethanolamine or glycine, etc) with 0.05-1% (w/v) blocking molecule. Wash, and resuspend in storage buffer to desired concentration : often 10 mg/ml. Storage buffer: pH 7-7.5 with 0.01-0.1% (w/v) blocking molecule and NaN3 (0.09%).

8°) Store at 4°c until used.

We supply OH-modified microspheres packaged at 10% or 1% solids in water.

Should you need additional information regarding our OH-modified Microspheres, please contact us.