CHROMATOGRAPHY General Protocol 1. 2. 3. 4. 5.

Prepare Column () Apply Sample Wash Elute Collect and Analyze Fractions adsorption ion exchange hydrophobic gel filtration affinity

Hydrophobic Interaction Chromatography (HIC) separates proteins (solutes) based on differences in hydrophobicity absorb and elute proteins from hydrophobic matrix (eg., octyl agarose, phenyl agarose) high salt promotes hydrophobic interactions salts vary in 'salting-out' effect and 'chaotropic' effect increasing salting-out effect PO4, SO4, Cl, Br, NO3, ClO4, I, SCN NH4, Rb, K, Na, Li, Mg, Ca, Ba increasing chaotropic effect

anions: cations:

elute under conditions which lower affect hydrophobic interactions lower salt concentration affect the structure of water (eg., decrease polarity, chaotropic agents) compete with detergents

Reverse Phase Chromatography carried out under denaturing conditions separates according to differences in the total hydrophobicity separation of small polypeptides and proteolytic fragments are common applications HIC vs. RPC
Mobile Phase Conditions Solute Properties Hydrophobic Polar Solvent Native Surface Residues Reverse Phase Nonpolar Solvent Denatured Total Residues

Gel Filtration Chromatography separates solutes based on molecular size also called molecular sieve chromatography or size exclusion chromatography chromatography media composed of cross-linked polymers degree of cross-linking will determine a pore size of matrix interaction of solute with matrix is determined by pore size larger molecules are excluded and thus migrate faster smaller molecules are included and thus migrate slower practical aspects choose media with desired separation range solute should not absorb to media (include salt to minimize ion exchange) resolution depends on small sample volume elution carried out in one column volume applications purification (limited resolution) desalting (as alternative to dialysis) size determination (stokes radius)

Size Calculation gel filtration columns are calibrated by using molecular weight standards mobility is determined from the following equation: Kav = Ve - Vo/Vt - Vo. void volume (Vo), also called the excluded volume, is the elution volume of a substance which is two large to enter the matrix of the support medium total volume (Vt) is calculated from the volume of the column bed (r2 x length) elution volume (Ve) is determined from column Kav values are plotted against the log of the molecular weight for each protein standard

Affinity Chromatography based on specific binding of protein to ligand ligands can include: substrate analogs, inhibitors, natural and artificial ligands, co-factors, metals, binding proteins, antibodies, etc Column preparation ligand attached to matrix (eg, CNBr-activated sepharose) linker arms matrix should not absorb contaminants covalent attachment of ligand should not alter binding properties binding should be specific, but affinity not so high as to prevent elution Elution: destabilize binding compete with free ligand change pH, ionic strength chaotropic or denaturing agents