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Star Republic: Guide for Biologists

Coupling to biotin

1. Change buffer for the protein (or antibody) solution to 0.1 M sodium carbonate.

2. Protein concentration should be 1-2 mg/ml.

3. Prepare fresh N-hydroxysuccinimido-Biotin (NHS-Biotin, 10 mg/ml) in anhydrous DMSO. NHS-Biotin attachs through free amino group, usually lysine residues.

4. For each mg of protein, add 20-100 mg of NHSE.

5. Incubate at room temperature in the dark for 3-4 hours with rotation.

6. Stop reaction by adding 2 ml of NH4Cl or by change buffer to 10 mM Tris-HCl, 150 mM NaCl, pH7.5-8.0.

7. If adding NH4Cl, an amine-free buffer (e.g., PBS) can be exchanged into for storage.