To improve the purification and to avoid as much as possible contaminating immunoglobulin chains, the IgG beads (IgG-Sepharose. Fast Flow. Amersham-Pharmacia (17-0969-01)) were crosslinked. A standard protocol as described by Harlow et al.1 was used.

Solutions:

0,2 M Sodium Borat (pH 9)

0,2 M Ethanolamine (pH 8

1 M NaCl / 50 mM Tris (pH 8)

0.01 % sodium azide in PBS

PBS

Cross-linking reagent: di-methylpimelimidate (solid) (Sigma, D8388)

Protocol:

1. Wash the IgG beads twice with 10 volumes of 0.2 M sodium borate

2. Centrifuge for 5 min at 3000 g.

3. Resuspend the beads in volumes of 0.2 sodium borate and add enough di-methylpimelimidate to bring the final concentration to 20 mM.

4. Mix for 30 min at room temperature on a shaker.

5. Stop the reaction by washing the beads twice in 0.2 M ethanolamine and incubate for 2 hours at room temperature in 0.2 M ethanolamine on a shaker.

6. Wash once in 10 volumes of the beads in 1 M NaCl / 50 mM Tris and incubate for 30 min in 10 volumes of the beads in 1 M NaCl / 50 mM Tris at room temperature.

7. Wash twice in 10 volumes PBS

8. Wash once in 10 volumes 0.01 % sodium azide in PBS

9. Store the beads in 0.01 % sodium azide.

10. Test a sample of the beads with SDS-sample buffer on a silver gel and compare them to beads, which are not cross-linked.