Science Gateway > Protocols > Cell Biology Protocols - Table of Contents
Star Republic: Guide for Biologists

Coupling of antibody to protein A or protein G beads

1. Choose the appropriate beads for your antibody.

2. Adjust pH of antibody solution to pH 8.0.

3. Wash protein-A(G)/agarose beads with 100 mM Tris-HCl, 0.5 M NaCl (pH 8.0).

4. Mix beads and antibody (2 mg antibody per ml of beads).

5. Incubate at room temperature for 1-3 hours with shaking.

6. Wash the beads 3 times with 10 volumes of 100 mM sodium borate pH 9.0.

7. Add 3 volumes fresh crosslinking solution (20 mM dimethyl pimelimidate (DMP) in 100 mM sodium borate pH 9.0).

6. Incubate at room temperature for 30-60 minutes on a shaking platform.

7. Remove crosslinking solution and terminate by adding 10 ml of 0.2 M ethanolamine-HCl pH 8.2.

8. Incubate at room temperature for 30-60 minutes on a shaking platform.

9. Wash twice for 3 minutes with 10 ml PBS/0.05% sodium azide. Store at 4 C. Alternatively, the beads can be transferred to a column, and stored in PBS/0.05% sodium azide at 4 C.

10. To check for crosslinking, remove 10 ml of beads before and after cross-linking, analysis on a 8% SDS-PAGE gel.