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Protein sequences and purification
• Primary struct ure diversity
– Proteins differ in sequence, size, charge and solubility
• Purification
– Bulk methods, chromatography, electrophoresis,
centrifugation
• Sequencing
– Peptide cleavage, Edman degradation, mass
spectrosopy
• Evolution
– Sequence relationships between proteins
Gel Filtration (Size exclusion)
• Column matrix chosen to
minimize interactions.
• Porous Beads occupy most of the
volume of the column.
• Pore size distribution presents
more space for smaller proteins.
• Large proteins see smaller
volume - elute earlier.
• Small proteins (also buffers,
salts) visit more nooks and
crannies and elute later.
Gel Electrophoresis
Principles
Force = Charge times Voltage
Velocity = Force / Resistance
Resistance is provided by the gel network, ()
Resistance increases with protein size, polyacrylamide
concentration
Cathode (negative termi nal, attracts cations)
Source of electrons, becomes basic
2H+ + 2e- –> H2
Anode (positive termi nal, attracts anions)
Sink for electrons, becomes acidic
H2O –> 4H+ + 4e- + O2
Buffers required to maintain pH
