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Star Republic: Guide for Biologists

Coupling of peptides to Sepharose beads

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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.