Affinity Chromatography - Department of Molecular and Cellular ...

Chapter 5
Designing affinity media using pre-activated matrices............................................ 100
Choosing the matrix ............................................................................................................................... 100
Choosing the ligand and spacer arm ......................................................................................................... 100
Choosing the coupling method ................................................................................................................. 100
Coupling the ligand ................................................................................................................................ 102
Binding capacity, ligand density and coupling efficiency ............................................................................ 103
Binding and elution conditions ................................................................................................................ 104
Coupling through the primary amine of a ligand .......................................................... 105
HiTrap NHS-activated HP, NHS-activated Sepharose 4 Fast Flow ................................................................ 105
CNBr-activated Sepharose ....................................................................................................................... 108
Immunoaffinity chromatography .............................................................................................................. 112
Coupling small ligands through amino or carboxyl groups
via a spacer arm ...................................................................................................... 113
EAH Sepharose 4B and ECH Sepharose 4B .............................................................................................. 113
Coupling through hydroxy, amino or thiol groups
via a 12-carbon spacer arm ...................................................................................... 116
Epoxy-activated Sepharose 6B ................................................................................................................. 116
Coupling through a thiol group .................................................................................. 120
Thiopropyl Sepharose 6B ........................................................................................................................ 120
Coupling other functional groups ............................................................................... 121
Chapter 6
Affinity chromatography and CIPP ........................................................................... 123
Applying CIPP .......................................................................................................... 124
Selection and combination of purification techniques.................................................. 124
Appendix 1 .......................................................................................................... 129
Sample preparation ................................................................................................. 129
Sample stability ..................................................................................................................................... 129
Sample clarification ............................................................................................................................... 130
Specific sample preparation steps ............................................................................. 131
Resolubilization of protein precipitates ..................................................................................................... 133
Buffer exchange and desalting .................................................................................. 134
Removal of lipoproteins ............................................................................................ 137
Removal of phenol red ............................................................................................. 137
Removal of low molecular weight contaminants .......................................................... 137
Appendix 2 .......................................................................................................... 139
Selection of purification equipment ........................................................................... 139
Appendix 3 .......................................................................................................... 140
Column packing and preparation ............................................................................... 140