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Star Republic: Guide for Biologists

Coupling of GST-fusion to glutathione beads

1. Purify GST-fusion protein without the elution step.

2. Transfer 1 ml protein/beads to a fresh tube.

3. Wash the protein/beads 3 times with 10 ml of 200 mM HEPES (pH 8.5).

4. Add 3 ml fresh crosslinking solution (20 mM dimethyl pimelimidate (DMP) in 200 mM HEPES pH 8.5).

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

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

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

8. Wash twice for 3 minutes with 5 ml of 150 mM NaCl, 200 mM Glycine-HCl (pH 2.0).

9. Wash twice for 3 minutes with 10 ml TBS (25 mM Tris (pH 7.5), 150 mM NaCl )/0.05% sodium azide. Store at 4 C. Alternatively, the beads can be transferred to a column, and stored in TBS/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.