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.
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