Proteins: Purification and Characterization

Key Concepts
• Proteins can be purified by various methods, mainly chromatographic, given:
o a source of the protein
o a detection method specific for the protein of interest

• Progress of purification can be followed using a purification table to monitor

o total protein (need a method to measure protein concentration)
o total activity (or other specific property) of protein of interest
o Specific activity (total activity/total protein), which indicates relative degree of purity;
constant specific activity is one indication that protein may be pure.

• Separation methods based on:

o differential centrifugation (to separate soluble from particulate fractions of crude cell
lysate)
o solubility (fractional precipitation, "salting out")
o column chromatography
 gel filtration (= size exclusion chromatog. = molecular sieve chromatog.), based on
size and shape
 ion exchange chromatography (based on net charge of protein at the working pH)
 affinity chromatography (based on specific ligand binding)

• Protein characterization:
o molecular weight
 electrophoresis (SDS-PAGE --> individual polypeptide chain molecular weights)
 gel filtration (calibrated column --> approx. native molecular weight if column run
under nondenaturing conditions)
 ultracentrifugation (depends on size and shape, but can give very accurate molecular
weight)
o isoelectric point (charge properties)
 isoelectric focusing (often used as the first dimension in 2-D gel separations to look at
ALL the proteins in a complex mixture)
o spectroscopic properties (give various kinds of structural and functional information)
 uv-visible spectroscopy
 absorbance spectroscopy
 fluorescence spectroscopy
 circular dichroism spectroscopy
 nmr spectroscopy
o determination of primary structure