Protein Purification and

Analysis
 Levels of Structure in Protein

• Primary: A description of all covalent bonds. The

sequence of AA residues
• Secondary: particularly stable arrangements of AA
giving rise to recurring structural patterns.
• Tertiary: All aspects of the 3D folding of a polypeptide.
• Quaternary: The spatial arrangement of multisubunits
protein
 Protein Purification
• Crude extract: breaking cells, by osmosis
lysis or homogenization.
• Fractionation: separate proteins into
different fraction based on size of charge.
• Salting out: The solubility of proteins is
lowered at high salt concentration.
Ammonium sulfate ((NH4)2SO4).
• Dialysis is a procedure to separate
proteins from solvents
Protein Purification: Column Chromatography
• The expansion of the
protein band in the mobile
phase is caused by
separation of proteins with
different properties and by
diffusional spreading. As
the length of the column
increases, the resolution
of two types of protein
improves.
• Rate is decreased and
resolution can decline
because of the diffusional
spreading
Ion-exchange Chromatography

• Cation exchangers
contain negatively
charged polymer
• Anion exchangers
contain positively
charged polymer.
• Is effected by pH
Size-Exclusion Chromatography
• Also called gel
filtration: The column
matrix is a cross-
linked polymer with
pores of selected size.
• Larger protein migrate
faster than smaller
ones because they
are too large to enter
the pores
Affinity Chromatography
• Separate protein by
their binding
specificities. The
proteins retained on
the column are those
that bind specifically to
a ligand cross-linked to
the beads. Proteins
that do not binds to
ligands are washed
through to column
Electrophoresis
• Separation of porteins is
based on the migration of
charged protein in an electric
field
• The migration of a protein in a
gel during electrophoresis is a
function of its size and shape
µ = V/E = Z/ f
M: electrophoretic mobility
V: velocity; E: electrical potential
Z: net charge; f: frictional
coefficient
SDS-PAGE: Sodium Dodecyl Sulfate (SDS)
Polyacrylamide Gel Electrophoresis

• SDS binds to most proteins probably by

hydrophobic interaction. One SDS for every two
AAs, Thus, each protein has a similar charge-
to-mass ratio.
• Coomassie blue stains protein. Western blot
Estimating the Molecular Weight of a Protein
Isoelectric Focusing
• pI of a protein: net
charge=0
• A pH gradient is
established by
allowing a mixture of
organic acids and
bases (ampholytes).
Protein migrates until
it reaches the pH that
matches its pI
Two-Dimensional Electrophoresis

• Separates proteins
of identical MW
that differ in pI or
proteins with
similar pI but
different MW.
Activity Vs. Specific Activity

• Unit: amount of
enzyme causing
transformation of 1
µ mole of substrate
per min. at 25 oC
under optimal
conditions
• Activity: Total units of
enzyme (U).
• Specific activity:
(U/mg) of total protein
Bacterial expression vectors
Bacterial expression vectors
Mammalian expression vector