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1. Swell 1 g CNBr-Activated Sepharose 4B beads in 50 ml 1 mM HCl for 15 minutes at room temperature.
2. Let the beads settle or spin down, remove supernatant.
3. Wash with 50 ml 1 mM HCl. Repeat twice.
4. Dissolve 10 mg peptides (or proteins, antibodies) in 5 ml
of the coupling buffer ( 0.1 M NaHCO3 pH 8.3, 0.5 M NaCl).
Buffer change may be necessary for proteins and antibodies.
5. Wash the beads with 10 ml of coupling buffer.
6. Remove supernatant. Add peptide solutions. Mix for 2 hours at room temperature
or overnight at 4 °C).
7. Remove supernatant. Add ethanolamine blocking reagent (6% (v/v) ethanolamine in the coupling buffer).
Mix for 2 hours at room temperature
or overnight at 4 °C).
8. Wash the beads with 0.1 M sodium acetate pH 4.0, 0.5M NaCl.
9. Wash the beads with 0.1 M Tris-HCl pH 8.0, 0.5M NaCl.
10. Wash the beads with PBS.
11. Change the buffer to PBS/0.02% Sodium Azide. Store at 4 °C.
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