Protein Purification

Ammonium sulfate precipitation

• uses differences in proteins' relative solubility in water and salt solutions
• AS is highly soluble, solutbility not very temp-dependeent, precipitated proteins retain activity when resolubilised
• low [AS] to remove impurities, then high [AS] (90%) to ppt proteins

Dialysis

• remove salts or other small molecules from solutions of proteins
• molecular weight cutoff (MWCO)
• place protein solution in dialysis bag, seal with space in bag, rinse outside, put in dialysis buffer, change buffer during dialysis

Ion Exchange Chromatography

• DEAE sepharose (agarose beads with diethylaminoethyl (-OCH2CH2N+(C2H5)2)groups attached): anion exchanger, binds negative proteins
• CM: cation exchanger
• elute by changing pH to pI of specific protein

Size Exclusion Chromatography (Gel Filtration)

• sephadex column: separate molecules of different sizes, cross-linked dextran polyers with different pore sizes
• bigger molecules move faster
• 2 markers: Blue Dextran (MW: 2E6)(V0), Orange-G (MW: 452)(Vt)
• separation depends on volume of sample, length of column, speed of elution, diameter of beads
• interference by aromatic substances and +ve ions having affinity for sephadex

Affinity

• affinity to ligand
• Ni-NTA resin column for a His-tagged protein, elute with imidazole or pH or EDTA which chelates Ni2+ and removes it with proteins from column

Protein Concentration Determination

1. Lowry Assay: destroys proteins, moderate variation with aa composition, slow, sensitive to salts and detergents
2. A280: not destructive, large variation with aa composition (measures absorbance of aromatic aa, WYF), immediate, sensitive to DNA/RNA
3. Dye binding (Coomassie Brilliant Blue)(Bradford assay): destructive, moderate variation with aa composition, 5-30min, sensitive to salts and detergents, absorbs at 595nm