Reagents

• pH 10 Coupling Buffer is 0.1M Sodium Citrate, 0.05M Sodium Carbonate pH 10.0
• Quenching Buffer is 1M Tris pH 7.4
• Wash Solution is 1M NaCl
• Reducing Agent is 5M Sodium CyanoBoroHydrate - TOXIC

Sample Preparation

• Have all reagents at room temperature
• Protein to be coupled cannot be in Tris (or other buffer with amines)
• Dialyze or dilute (1:3) protein into Coupling buffer, 1-20mg/ml desired
• Pack gel into columns, without top disc
• Allow storage buffer to pass through, but never allow gel bed to dry

Ligand Addition

• Equilibrate column wiht 2.5 volumes coupling buffer, drain
• Add 1-2 volumes of ligand solution
• Cap column and mix by end-over-end rocking 4 hours
• Drain then wash with 2.5 volumes coupling buffer

Ligand Coupling

• In Fume Hood Add 1 volume coupling buffer and 0.02 volumes Reducing Agent
• Cap column and mix by end-over-end rocking 4 hours to overnight

Quench Unused Sites

• Wash with 2 volumes Quenching Buffer, drain
• In Fume Hood Add 1 volume Quenching buffer and 0.02 volumes Reducing Agent
• Cap column and mix by end-over-end rocking 30 minutes
• Wash with 10 volumes Wash Solution

Storage

• Push polyethylene disc to 1mm above gel surface
• Store in buffer in dark at 4°C with 0.05% Sodium Azide