PROTEIN ANALYSIS

CHAPTER2, Medical Biochemistry

Biochemical Methods to Analyze
Proteins
• Electrophoresis
• Chromatography: Gel filtration, ion
exchange, affinity
• Mass Spectrometry, X-ray
Crystallography, NMR
 Protein Separation by SDS-
Polyacrylamide Gel Electrophoresis
Presence of SDS, a detergent,
denatures and linearizes a protein
(Na and sulfate bind to charged
amino acids, the hydrocarbon chain
interacts with hydrophobic residues).
An applied electric field leads to
separation of proteins based on size
through a defined gel pore matrix.

For electrophoresis in the absence

of SDS, separation is based on size,
charge and shape of the protein
(proteins are not denatured and can
potentially retain function or activity)
SDS-Polyacrylamide Gel (cont)
Separation of proteins
based on their size is
linear in relation to the
distance migrated in the
gel. Using protein
standards of known
mass and staining of the
separated proteins with
dye, the mass of the
proteins in the sample
can be determined. This
is useful for purification
and diagnostic purposes.
 Gel filtration
Separation is based on protein size.
Dextran or polyacrylamide beads of
uniform diameter are manufactured
with different pore sizes. Depending
on the sizes of the proteins to be
separated, they will enter the pore if
small enough, or be excluded if they
are too large.

Hydrophobic Chromatography
Proteins are separated based on their
net content of hydrophobic amino
acids. A hydrocarbon chain of 4-16
carbons is the usual type of resin.
Ion Exchange Chromatography
Separation of proteins based on
the net charge of their constituent
amino acids. Different salt
concentrations can be used to elute
the bound proteins into tubes in a
fraction collector. As shown below,
resins for binding (+) or (-) charged
proteins can be used
 Affinity Chromatography
• Based on the target proteins ability to bind a
specific ligand, only proteins that bind to this
ligand will be retained on the column bead. This is
especially useful for immunoaffinity purification
of proteins using specific antibodies for them.
• Example:
 Protein Structure Methods
• The sequence of a protein (or peptide) is
determined using sophisticated Mass
Spectrometry procedures. The three
dimensional structures of proteins are
determined using X-ray crystallographic and
NMR (nuclear magnetic resonance)
spectroscopic methods.
• Protein sequence data banks useful for
structural and sequence comparisons
• Please note that the new discipline termed
“Proteomics” is evolving to incorporate cross-
over analysis of sequence data banks, Mass
Spec methodology, and living cells