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1. Wash particles (e.g., 100 mg of 1 μm carboxylated latex beads) into coupling buffer (50 mM MES, pH 6.0). Suspend in 5 ml coupling buffer. The addition of a dilute detergent solution may be done to increase bead stability and prevent clumping (e.g., 0.01 percent SDS). Avoid the addition of any components containing carboxylates or amines (such as acetate, glycine, Tris, imidazole, etc.). Also, avoid the presence of thiols (e.g., DTT, 2-mercaptoethanol, etc.), as these will react with EDC and effectively inactivate it.马尔斯
2. Add 100 mg of EDC and 100 mg of sulfo-NHS. Mix to dissolve. To facilitate faster dissolution, EDC and sulfo-NHS may be dissolved immediately before use as a concentrated stock solution in reaction buffer and then an aliquot of this solution added to the particle suspension to obtain the correct ?nal concentration.
3. React for 15 minutes at room temperature.
4. Quickly wash beads 2 times with coupling buffer using centrifugation and resuspend using a sonic probe in 5 ml of the same buffer.博弈清末
Coupling
5. Dissolve protein to be coupled in 5 ml coupling buffer at a concen tration suf? cient to provide 1- to 10-fold molar excess of ligand over the maximal calculated monolayer concentration for the amount and type of beads used. For particle manufacturers reporting a carboxylate concentration in meq/g, this is equivalent to μmol/mg. The optimal protein concentration should be optimized. Note: Too low a protein concentration may result in particle crosslinking. For coupling of expensive antibodies that may not be available in enough quantity to reach the optimal molar ratio on the particles, the addition of another protein (i.e., bovine gamma globulin or BSA) may be done to take up remaining reactive sites.
6. Combine the protein solution with particles and mix thoroughly.
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7. React at room temperature 2–4 hours with mixing.
8. Wash beads with coupling buffer and resuspend in the same buffer containing 100 mM of an amine-containing hydrophilic quenching molecule to block excess reactive sites (i.e., ethanolamine or Tris).年降水量
9. Wash beads and resuspend in an appropriate buffer for storage.
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