Unit 6

PROTEIN PURIFICATION AND

CHARACTERIZATION
A. Extraction of proteins from cells
B. Column chromatography
1. Size-exclusion chromatography
2. Affinity chromatography
3. Ion-exchange chromatography
4. High-performance liquid chromatography
C. Electrophoresis
1. SDS-PAGE
2. Isoelectric focusing
D. Determining primary structure of proteins

Prepared by: RJ A. Sorgon, MSc, MLS, CLT

Learning Outcomes
At the end of this unit, the students are expected to:
1. Identify and describe the different techniques used to extract and purify
proteins
2. Describe the process of differential centrifugation, salting out with
ammonium sulfate and dialysis in protein purification
3. Discuss the principles of the following techniques
a. ion-exchange chromatography
b. size-exclusion chromatography
c. affinity chromatography
d. high-performance liquid chromatography
e. SDS-PAGE
f. isoelectric focusing
4. Describe migration of serum proteins
5. Describe the techniques in determining amino acid sequences of a primary
structure of protein
Working with proteins
• To study a protein in detail, the researcher must be able to
separate it from other proteins in pure form and must have the
techniques to determine its properties.

General Steps
1. selection of sample
2. solubilization of protein from sample
3. purification of protein of interest from homogenized sample
4. characterization of protein of interest
Overview of protein purification
and characterization
Protein Extraction
• The source of a protein is generally tissue or microbial cells.
• The first step in protein purification is to break open the cells (cell lysis),
releasing their proteins into a solution called a crude extract.
Protein Extraction
• Cell lysis methods can be divided into two main categories:
a. reagent-based
b. physical disruption
• Homogenization – process of rupturing the plasma membrane (includes
bacterial/plant cell wall) to release the protein from the cell

Image from Thermofischer

Physical methods of cell lysis
Lysis method Apparatus Description
Mechanical Waring blender Rotating blades grind and
Polytron disperse cells and tissues
Liquid Dounce Cell or tissue suspensions are
Homogenization homogenizer sheared by forcing them through
a narrow space

Sonication Sonicator High frequency sound waves

shear cells
Freeze-thaw Freezer or dry Repeated cycles of freezing and
ice with ethanol thawing disrupt cells through ice
crystal formation

Manual grinding Mortar and Grinding plant tissue, frozen

pestle in liquid nitrogen
Protein purification
• Highly purified protein is essential for the detailed examination of
its physical and functional properties.
• Methods for separating proteins take advantage of properties that
vary from one protein to the next, including:
• size
• charge
• binding properties
• Classic approaches exploit differences in relative solubility of
individual proteins such as
• pH (isoelectric precipitation)
• polarity (precipitation with ethanol or acetone)
• salt concentration (salting out with ammonium sulfate)
Differential centrifugation
• Differential centrifugation can be used to prepare subcellular
fractions or to isolate specific organelles.
• The extract is then subjected to treatments that separate the
proteins into different fractions based on a property such as size
or charge, a process referred to as fractionation.
Salting out
• The solubility of proteins is generally lowered at high salt
concentrations, an effect called salting out.
• The addition of certain salts in the right amount can selectively
precipitate some proteins, while others remain in solution.
• Ammonium sulfate ((NH4)2SO4) is often used to salt out proteins.
• The precipitated proteins are removed from those remaining in
solution by low-speed centrifugation.
Dialysis
• A solution containing the protein of interest usually must be further
altered before subsequent purification steps are possible.
• Dialysis is a procedure that separates proteins from small solutes
by taking advantage of the proteins’ larger size.
• The partially purified extract is placed in a bag or tube made of a
semipermeable membrane.
• The membrane allows the exchange of salt and buffer but not
proteins.
• Dialysis retains large proteins within the membranous bag or tube
while allowing the concentration of other solutes in the protein
preparation to change until they come into equilibrium with the
solution outside the membrane
Dialysis
Chromatography
• Chromatographic separations partition molecules between two
phases:
• mobile phase
• stationary phase
• For separation of amino acids or sugars, the stationary phase, or
matrix, may be:
• a sheet of filter paper (paper chromatography)
• thin layer of cellulose, silica, or alumina (thin-layer
chromatography [TLC]).
• The most powerful methods for fractionating proteins make use of
column chromatography.
Column chromatography
• A porous solid material with appropriate chemical properties (the
stationary phase) is held in a column, and a buffered solution (the
mobile phase) percolates through it.
• The protein-containing solution, layered on the top of the column,
percolates through the solid matrix as an expanding band within
the larger mobile phase.

Column chromatography
1. Ion-exchange chromatography
2. Size-exclusion chromatography
3. Affinity chromatography
4. High-performance liquid chromatography
Column
chromatography
• These stationary phase matrices
interact with proteins based on
their charge, hydrophobicity, and
ligand-binding properties.
• A protein mixture is applied to
the column and the liquid mobile
phase is percolated through it.
• Individual proteins migrate faster
or more slowly through the
column depending on their
properties.
• Small portions of the mobile
phase or eluant are collected as
they emerge.

Image from Nelson and Cox (2017)

Ion-exchange chromatography
• exploits differences in the net electric charge of proteins at a
given pH
• The column matrix is a synthetic polymer (resin) containing
bound charged groups
• cation exchangers – with bound anionic groups
• anion exchangers – with bound cationic groups

• The affinity of each protein for the charged groups on the column
is affected by the pH and the concentration of competing free salt
ions in the surrounding solution.
• Separation can be optimized by gradually changing the pH
and/or salt concentration of the mobile phase so as to create a
pH or salt gradient.
Ion-exchange
chromatography
• proteins interact with the
stationary phase by charge to
charge interactions
• Proteins with a net positive
charge at a given pH adhere
to beads with negatively
charged functional groups
such as carboxylates or
sulfates (cation exchangers).
• Similarly, proteins with a net
negative charge adhere to
beads with positively charged
functional groups, typically
tertiary or quaternary amines
(anion exchangers).

Image from Nelson and Cox (2017)

Size-exclusion
chromatography
• also called gel filtration
chromatography

• separates proteins according

to size, where large proteins
emerge from the column
sooner than small ones

• The solid phase consists of

cross-linked polymer beads
with pores or cavities of a
particular size.

Image from Nelson and Cox (2017)

 Size-exclusion chromatography

• Large proteins cannot enter the cavities and so take a short (and
rapid) path through the column, around the beads.
• Small proteins enter the cavities and are slowed by their more
labyrinthine path through the column.
Image from Rodwell et al (2018)
Affinity
chromatography
• based on binding affinity (high
selectivity of most proteins for
their ligands)
• The beads in the column have a
covalently attached chemical
group called a ligand (a group or
molecule that binds to a
macromolecule such as a protein)
• When a protein mixture is added
to the column, any protein with
affinity for this ligand binds to the
beads, and its migration through
the matrix is retarded.

Image from Nelson and Cox (2017)

Affinity chromatography
• For example, if the biological function of a protein involves binding to ATP,
then attaching ATP to the beads in the column creates an affinity matrix
that can help purify the protein.
• As the protein solution moves through the column, ATP-binding proteins
(including the protein of interest) bind to the matrix.
• After proteins that do not bind are washed through the column, the bound
protein is eluted by a solution containing either a high concentration of
salt or free ligand—in this case, ATP.
• Salt weakens the binding of the protein to the immobilized ligand,
interfering with ionic interactions.
• Free ligand competes with the ligand attached to the beads, releasing the
protein from the matrix; the protein product that elutes from the column is
often bound to the ligand used to elute it.
High-performance Liquid
Chromatography
• HPLC employs incompressible silica or alumina microbeads as
the stationary phase and pressures of up to a few thousand psi.
• Incompressible matrices permit both high flow rates and
enhanced resolution.
• HPLC can resolve complex mixtures of lipids or peptides
whose properties differ only slightly.
• high-pressure pumps hasten the movement of the protein
molecules down the column
• By reducing the transit time on the column, HPLC can limit
diffusional spreading of protein bands and thus greatly improve
resolution.
Electrophoresis
• Another important technique for the separation of proteins is
electrophoresis, which is based on the migration of charged
proteins in an electric field
• These procedures are not generally used to purify proteins in
large amounts, because electrophoretic methods often adversely
affect the structure and thus the function of proteins.
• Proteins can be visualized and separated, permitting a quick
estimate of the number of different proteins in a mixture or the
degree of purity of a particular protein preparation.
• Electrophoresis allows determination of crucial properties of a
protein such as its isoelectric point and approximate molecular
weight.
Electrophoresis
• Electrophoresis is useful as an analytical method.
• separates charged biomolecules based on the rates at which they
migrate in an applied electrical field
• The migration of a protein in a gel during electrophoresis is
affected by its size and its shape.

• Electrophoresis of proteins is generally

carried out in gels made up of the cross-linked
polymer polyacrylamide.

• The polyacrylamide gel acts as a molecular

sieve, slowing the migration of proteins
approximately in proportion to their charge-to-
mass ratio.
Electrophoresis

Image from Nelson and Cox (2017)

Estimating the molecular weight of a
protein
• The electrophoretic mobility
of a protein on an SDS
polyacrylamide gel is related
to its molecular weight, Mr.
• Standard proteins of known
molecular weight are
subjected to electrophoresis
(lane 1).
• These marker proteins can
be used to estimate the
molecular weight of an
unknown protein (lane 2).

Image from Nelson and Cox (2017)

SDS-PAGE
• An electrophoretic method commonly employed for estimation of
purity and molecular weight makes use of the detergent sodium
dodecyl sulfate (SDS)
• SDS binds to most proteins in amounts roughly proportional to the
molecular weight of the protein
• The bound SDS contributes a large net negative charge,
rendering the intrinsic charge of the protein insignificant and
conferring on each protein a similar charge-to-mass ratio.
• SDS-PAGE separates proteins almost exclusively on the basis of
mass (molecular weight), with smaller polypeptides migrating
more rapidly.
SDS-PAGE
• SDS denatures and binds to proteins at a ratio of one molecule of
SDS per two peptide bonds.

• When used in conjunction with 2-mercaptoethanol or

dithiothreitol to reduce and break disulfide bonds.

• Individual polypeptides trapped in the acrylamide gel are

visualized by staining with dyes such as Coomassie blue, which
binds to proteins but not to the gel itself
Serum protein
electrophoresis
Hemoglobin electrophoresis
Isoelectric focusing
• This technique separates proteins according to their isoelectric
points.
• A stable pH gradient is established in the gel by the addition of
appropriate ampholytes.
• With an applied electric field, proteins enter the gel and migrate
until each reaches a pH equivalent to its pI.
• A pH gradient is established by allowing a mixture of low
molecular weight organic acids and bases (ampholytes) to
distribute themselves in an electric field generated across the gel.
• When a protein mixture is applied, each protein migrates until it
reaches the pH that matches its pI.
• Proteins with different isoelectric points are thus distributed
differently throughout the gel.
Isoelectric focusing

Image from Nelson and Cox (2017)

Two-dimensional
electrophoresis
• a process when isoelectric focusing and SDS electrophoresis
are sequentially combined
• permits the resolution of complex mixtures of proteins
• a more sensitive analytical method than either electrophoretic
method alone
• proteins of identical molecular weight that differ in pI, or
proteins with similar pI values but different molecular weights
Two-dimensional
electrophoresis
• proteins are first separated by
isoelectric focusing in a cylindrical
gel
• The gel is then laid horizontally on
a second, slab-shaped gel, and
the proteins are separated by
SDS-PAGE
• horizontal separation reflects
differences in pI
• vertical separation reflects
differences in molecular weight.

Image from Nelson and Cox (2017)

Protein Sequencing
Steps
1. Hydrolysis – effected by acid, alkali, or enzyme
a. acid hydrolysis – heating in presence of 6 M HCl at
110oC,10-100 h destroys Trp
b. alkaline hydrolysis – heating in presence of 4 M
NaOH; does not damage Trp but destroys a lot more
amino acids
c. enzymatic hydrolysis – use proteases/peptidases

2. Identification of the products of hydrolysis

3. Fitting the pieces together (as in a jigsaw puzzle)

Enzymatic hydrolysis
1. Exopeptidases – cleaves external peptide bonds
a. aminopeptidase – sequentially cleave peptide bonds
beginning at the N-terminal end
b. carboxypeptidase – sequentially cleave peptide bonds
beginning at the C-terminal end

2. Endopeptidases – cleaves internal peptide bonds

a. trypsin – cleaves peptide bonds at the carboxyl end of
two strongly basic amino acids, Arg and Lys
b. chymotrypsin – cleaves peptide bonds at the carboxyl end of
the three aromatic amino acids, Phe, Tyr, and Trp
c. pepsin – cleaves peptide bonds at the amino end of the
three aromatic amino acids, Phe, Tyr, and Trp
Enzymatic hydrolysis

• peptide bond cleavage occurs on either the carbonyl (C) or the

amino (N) side of the indicated amino acid residues.

Nelson and Cox (2017)

Sanger method
• Sanger reduced the disulfide bonds and cleaved each chain into
smaller peptides using trypsin, chymotrypsin, and pepsin.
• The resulting peptides were then isolated and treated with acid to
hydrolyze peptide bonds and generate peptides with as few as
two or three amino acids.
• Each peptide was reacted with 1-fluoro-2,4-dinitrobenzene
(FDNB, Sanger’s reagent), which derivatizes the exposed α-amino
groups of the amino-terminal residues.
• The amino acid content of each peptide was then determined and
the amino-terminal amino acid identified.
• Sanger determined the complete sequence of insulin (awarded a
Nobel Prize in 1958)
Edman Degradation
• Pehr Edman introduced phenylisothiocyanate (PITC, Edman’s
reagent) to selectively label the amino-terminal residue of a
peptide
• Successive rounds of derivatization with Edman’s reagent can be
used to sequence many residues of a single sample of peptide..
• Following cleavage, the resulting peptides are purified by
reversed-phase HPLC and sequenced.
Steps in sequencing a
polypeptide

Image from Nelson and Cox (2017)

Sequencing Large Proteins
into Smaller Segments
• large polypeptides found in proteins must be broken down into
smaller pieces to be sequenced efficiently
• the protein is cleaved into a set of specific fragments by chemical or
enzymatic methods

Steps
1. breaking disulfide bonds
2. cleaving the polypeptide chain – proteases catalyze the hydrolytic
cleavage of peptide bonds
3. sequencing the peptides each peptide fragment resulting from the
action of trypsin is sequenced separately by the Edman procedure.
4. ordering the peptide fragments
5. locating disulfide bonds
Mass spectrometry
• A technique used to quantify known materials, to identify unknown
compounds, and to determine the structure and chemical
properties of different molecules.
• This technique basically studies the effect of ionizing energy on
molecules.
• A mass spectrometer generates multiple ions from the sample
under investigation, it then separates them according to their
specific mass-to-charge ratio (m/z), and then records the relative
abundance of each ion type.
• Mass Spectrometry allows precise determination of the molecular
mass of peptides
• This information can be used for protein identification, de novo
sequencing, and identification of post-translational modifications
Proteomics and the Proteome
• Proteomics refers to the analysis of complete protein content in a
living system, including co- and post-translationally modified
proteins and alternatively spliced variants.
• Scientists are now trying to determine the primary sequence and
functional role of every protein expressed in a living cell, known as
its proteome.

• A major goal is the identification of proteins and of their

posttranslational modifications whose appearance or
disappearance correlates with:
• physiologic phenomena
• aging
• specific diseases
References
1. McKee, T., and McKee, J. R. (2012). Biochemistry: The
molecular basis of life. Oxford: Oxford University Press.
2. Nelson, D. L., and Cox, M. M. (2017). Lehninger Principles of
Biochemistry. Seventh edition. New York: Worth Publishers.
3. Pratt, C. and Cornely, K. (2014). Essential Biochemistry. Third
edition. USA: John Wiley and Sons.
4. Rodwell, V. et al. (2018). Harper's illustrated biochemistry. 31st
edition. New York: McGraw-Hill.
5. Stoker, S. (2015). General, Organic, and Biological Chemistry.
Sixth Edition. United States of America: Cengage Learning.