Affinity chromatography in practice

Experimental affinity chromatography
	Mono-specific	Group-specific	Fusion protein
Affinity chromatography medium	Media for affinity chromatography should be highly porous to guarantee high coupling yields and full access to the affinity ligand.	Media are commercially available in prepacked columns and kits to guarantee proper function.	Media are commercially available in prepacked columns and kits to guarantee proper function.
Column	Affinity chromatography is step-eluted. Columns are therefore typically "short and fat".	Affinity chromatography is step-eluted. Columns are therefore typically "short and fat".	Columns are typically "short and fat" and in HiTrap™ format. For parallel purification use spin columns.
Column regeneration and storage .	Some affinity ligands are sensitive to proteases. Follow recommended regeneration and storage procedures to ensure full column lifetime.	Some affinity ligands are sensitive to proteases. Follow recommended regeneration and storage procedures to ensure full column lifetime.	Some affinity ligands are sensitive to proteases. Follow recommended regeneration and storage procedures to ensure column lifetime. Re-use of columns should be restricted to identical fusion proteins to prevent cross contamination.
Eluents	Harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column after use.	Harsh elution conditions, which may influence ligand lifetime are sometimes required. Always wash column after use.	Columns intended for His-tagged proteins may require to be re-loaded with the metal ion used.
Flow rates	Binding/desorption kinetics may restrict applicable flow rate.	Binding/desorption kinetics may restrict applicable flow rate.	For proper function follow recommended procedure.
Sample volume	Sample volume is generally of less importance, as long as the maximum load capacity of the gel is not voilated Samples too dilute may hamper binding if KD is higher than optimal.	Sample volume is generally of less importance, as long as the maximum load capacity of the gel is not voilated.	Sample is generally of less importance, as long as the maximum load capacity of the gel is not voilated.

• Filter or spin sample to remove particulate matter.
• Use a desalting column to match the sample conditions to those of the binding buffer.

• Collect eluted fractions in small amounts of concentrated buffer of a pH within the stability window of the target protein.
• Immediately after elusion, use a desalting column to transfer the eluted target protein to gentle conditions.