3.

Protein Purification

Support materials: Tropp B.E, Molecular Biology Genes to Proteins 3rd Edition, Jones and Bartlett.
Lecture 4
 General step in purification
Aim of purification is to obtain purified product:
subsequent use of purified proteins 1) to study its
functions, properties (in research) 2) application in
manufacturing, health, food sectors.

2
 Purifying a protein requires a
strategy
1. Identify how to isolate the protein:
Isolation of a protein from the cell and into a solution: procedure
require mechanical disruption/organic solvent/detergent

2. Once a protein has been removed from its natural environment

these factors must be controlled or considered to avoid damage
and to keep proteins stable:

i) pH – suitable buffer for protein to avoid denaturation.

ii) temperature – know the thermal stability of the protein. 3

Protein purification normally carried out at low
temperature
Purifying a protein requires a
strategy - continue
iii) presence of degradative enzymes –destroying tissues
also release degradative enzymes e.g. proteases and
nucleases. Adjusting pH and temperature can inactivate the
enzymes (but not adversely affect protein of interest!).

iv) adsorption to surfaces-denatured by contact with air-water

interface or glass/plastic interface, therefore minimize
foaming and protein solution is kept concentrated.

v) long-term storage-processes such as slow oxidation and

microbial contamination must be prevented. Also store at -80
deg C to -196 deg C (temperature of liquid nitrogen) 4
Isolation of Proteins from
Cells
Many different proteins exists within one cell
• Many steps needed to extract protein of
interest, and separate from many
contaminants
• Before purification begins, protein must be
released from cell by homogenization

5
Isolation of Proteins from
tissues/cells
Protein extraction using these approaches:
▪ Mechanical/physical disruption: using
pastle and mortar, blender, homogenizer
▪ Chemical disruption: using salts, solvent,
liquid nitrogen (extreme temperature).

6
Salting Out

• After proteins solubilized, they can be purified based on solubility

(usually dependent on overall charge, ionic strength, polarity
• Ammonium sulfate (NH4SO4) commonly used to “salt out”

• Takes away water by interacting with it, makes protein less soluble
because hydrophobic interactions among proteins increases

• Different aliquots taken as function of salt concentration to get

closer to desired protein sample of interest (30, 40, 50, 75%
increments)
• One fraction has protein of interest/mixed of protein 7
Centrifugation
• Centrifuges have many applications, however they are
used primarily for:

1. The preparation of biological samples

2. Analysis of the physical properties of biomolecules,
organelles or cells – ex. Differential and density
gradient measurements

8
• It turns out that if:

• A particle/molecule is the same density as the liquid

around it, the particle doesn’t move

• A particle/molecule is more dense than the liquid, it

moves down the tube

• A particle/molecule is less dense than the liquid, it

moves up!
 Principle of Operation
• A centrifuge is a piece of equipment,
generally driven by an electric motor,
that puts an object in rotation around a
fixed axis, applying a force
perpendicular to the axis to separate
substances of different densities.
• Tubes in the centrifuge are tilted so
centrifugal force can pull denser
substances towards the bottom of the
tube.
• Relative Centrifugal Force (RCF)
measures acceleration applied to the
sample

https://rice360.wildapricot.org/Resources/
BIOE449/Lecture%203%20Centrifuge.ppt
FORCE IN A CENTRIFUGE IS PROPORTIONAL
TO TWO THINGS
• First, it depends on how fast the centrifuge spins

• Second, it depends on the radius of rotation –

think about “crack the whip”
The greater the radius of
rotation, the more force that is
experienced by the molecule
thus less dense molecules
sediments
SUPERNATANT AND A PELLET

• Supernatant is the liquid above a solid

residue (upper phase) after centrifugation
– low density molecules are found
• Pellet is particles that sediment at the
bottom of the centrifugation tube (lower
phase)
– high density molecules are found
 HOW FAST DOES A
PARTICLE SEDIMENT?
✓ Depends on:
• RCFs in the centrifuge – greater RCF, shorter time for
separation
• Size of particle – small size/ low molecular weight
molecules require longer time to sediment
• Particle density – low density molecules require longer
time - thus protein require longer time to sediment
• Liquid density – in high viscosity and density solution,
sedimentation of molecule is slow thus require longer
sedimentation time – this because the molecule
moves up
• Liquid viscosity
This is how separation is achieved

protein less
dense
Ex. 16S RNA
and 23S RNA
REMEMBER:
Separations are
due to the
different density
RNA more
dense of biomolecules

www.phys.sinica.edu.tw/TIGP.../AC_Chapter%203%20Centrifugation%200321.pdf
Differential
centrifugation

• Sample is spun, after lysis,

to separate unbroken cells,
nuclei, other organelles and
particles not soluble in
buffer used

• Different speeds of spin

allow for particle separation

16
3. Protein Purification
- Column chromatography

17

Support materials: Tropp B.E, Molecular Biology Genes to Proteins 3rd Edition, Jones and Bartlett.
Lecture 5
 Definition of chromatography

IUPAC definition (International Union of pure and applied

Chemistry) (1993):
Chromatography is a physical method of separation in which the
components to be separated are distributed between two phases,
one of which is stationary while the other moves in a definite
direction. (in mobile phase)

The stationary phase may be a solid, or a liquid supported on a

solid or gel, the mobile phase may be either a gas or a liquid.

Basics of Chromatography: https://www.cmscbe.com/.../BASICS%20OF%20CHROMATOGRAPHY.PPT

 Uses for Chromatography

Chromatography is used by scientists to:

•Analyze – examine a mixture, its components, and their
relations to one another
•Identify – determine the identity of a mixture or
components based on known components
•Purify – separate components in order to isolate one of
interest for further study
•Quantify – determine the amount of the a mixture
and/or the components present in the sample

Basics of Chromatography: https://www.cmscbe.com/.../BASICS%20OF%20CHROMATOGRAPHY.PPT

Mobile Phase – gas or liquid that carries the mixture of
components through the stationary phase.

Stationary Phase – the part of the apparatus that holds

the components as they move through it,
separating them.

Basics of Chromatography: https://www.cmscbe.com/.../BASICS%20OF%20CHROMATOGRAPHY.PPT

 Basis of Chromatography
• Technique for separating the components, or solutes, of a
mixture on the basis of the relative amounts of each
solute distributed between a moving fluid stream, called
the mobile phase, and a contiguous stationary phase.
The mobile phase may be either a liquid or a gas, while
the stationary phase is either a solid or a liquid.
• 2 phases:
• Stationary: solutes/samples/proteins interacts with this
phase
• Mobile: Flows over the stationary phase and carries
along with it the solutes (samples/proteins) to be
separated
21
 Column chromatography
• The stationary phase is packed into a glass or metal column.
• The stationary phase is either discrete small particles (the
matrix) and packed into the column or applied as a thin film to
the inside wall of the column.

• The mixture of analytes is then applied and the mobile phase,

referred as the eluent, is passed through the column either by
use of a pumping system or applied gas pressure.
• As the eluent flows through the column the analytes separate
on the basis of their distribution coefficients and emerge
individually in the eluate as it leaves the column.
 Column chromatography is use
to separate proteins

Adapted from an illustration by

Wilbur H. Campbell, Michigan
Technological University
(http://www.bio.mtu.edu/camp
bell/bl4820/lectures/lec6/482w
62.htm) Figure 2.11
 GEL FILTRATION

✓ Gel filtration separates molecules according to the differences

in size as they pass through the filtration medium packed in
the column.
✓ It is well suited for biomolecules that are sensitive to pH,
concentration and harsh environment.
✓ Parameters that affects gel filtration are, particle size, flow
rate, packaging density, porosity of the particle and viscosity
of the mobile phase.

Basics of Chromatography: https://www.cmscbe.com/.../BASICS%20OF%20CHROMATOGRAPHY.PPT

Size Exclusion/
Gel-filtration
(Cont’d)

25
 Size-Exclusion/Gel-Filtration
• Separates molecules based on molecular size.
• Stationary phase composed of cross-linked gel
particles.
• Extent of cross-linking can be controlled to
determine pore size
• The particles have pores, with different pores sizes
• Molecules that are smaller than the pore size can enter the
particles and therefore have a longer path and longer transit
time than larger molecules that cannot enter the particles
• Smaller molecules enter the pores and are delayed
in elution time. Larger molecules do not enter and
elute from column before smaller ones.

• Pore size – related to exclusion limit and fractionation

range
26
 Definition
Exclusion limit: defined as the molecular mass of
the smallest molecule that cannot diffuse into the
inner volume of the gel matrix.

e.g. Sephadex G-50 exclusion limit is 30 kDa;

molecules with mw greater than this value would
pass through the column without entering the gel
pores

Fractionation range: Sephadex G-50 has a

fractionation range of 1.5 to 30 kDa
 Loading vs Elution buffer

Loading buffer: Buffer/solution used to bind solutes

(for example, proteins) to stationary phase or
matrices/beads

Elution buffer: Buffer/solution used for elution

(desorption) of bound solutes (for example, proteins)
from a column.
 Size exclusion chromatography chromatogram
• LARGE Molecules
cannot enters pores
(gel/matrices), are
excluded from the
matrices (shortest
retention time) and
Absorption (nm)

elute first

• SMALL Molecules
enter the pores; are
retained and elute
later

• Molecules with
smallest molecular
weight enter the
matrix, has longest
retention time and
Elution volume or Retention time elute latest
 Physical characterization of gel
filtration chromatography
• Exclusion limit:
• Fractionation range:
• Water regain and bed volume: media are
often supplied in dehydrated form, must be
swollen in a solvent (usually water) before
use; the water taken up by 1 g of dry gel is
water regain – this cannot be used to estimate
final volume of packed gel column
- bed volume is the final volume taken up by 1
g dry gel when swollen in water/solvent
• Gel particle and size: Gel particles should be
spherical to provide uniform bed with a high
density of pores. Particle size is defined either
by mesh size or bead diameter (µm).
- The degree of resolution afforded by a column
and the flow rate depend on particle size
• Void volume: is the volume in total space
surrounding the gel particles in packed
volume – is determined by measuring the
volume of solvent required to elute a solute
that is excluded from the gel matrix –
calibration using dye, blue dextran (2 000 kDa)

• Elution volume: is the volume of eluting buffer

necessary to remove an analyte/molecule
from packed column or gel particles/matrices.
 REMEMBER
• In column chromatography, the total volume of
material, both solid and liquid, in the column;
i.e. the volume of the support particles plus the
void volume. It is synonymous with column
volume for a packed column.
Retention time:
It is the characteristic; time it takes for a particular
analyte (proteins) to pass through the system (from the
column inlet to the detector) under set conditions.

Column volume:
The geometrical volume of the chromatography bed.

Basics of Chromatography: https://www.cmscbe.com/.../BASICS%20OF%20CHROMATOGRAPHY.PPT

Size-exclusion chromatography

35
Size-exclusion chromatography
Determination of fractioned protein:
1) Absorbance (protein) at 280 nm is use to
identify protein-containing fractions.
2) You can also perform protein assay or
enzyme specific assay.

36
 Ion Exchange
• Proteins are separated on the basis of
their overall net charge (less specific
than affinity)
• Cation exchange chromatography
separation for positively charged
protein. Columns containing negatively
charged solid phase e.g.
carboxylmethyl cellulose
• Anion exchange chromatography –
separation for negatively charged
protein. Columns containing positively
37
charged solid phase e.g. DEAE
cellulose.
ION EXCHANGERS
• Cation exchangers (negative ions – stationary)
Separation for positively charged proteins
• Anion exchangers (positive ions - stationary)
Separation for negatively charged proteins

The most frequently used anion exchanger is a matrix with

attached diethylaminoethyl (DEAE) groups and for cation
exchanger is a matrix bearing carboxymethyl (CM) groups
 Steps in ion-
exchange

Handbook on Ion-exchange
chromatography: Principles and
method ( GE Healthcare online)
Elution of bound molecules
• Elution of bound molecules are achieved by increasing the
ionic strength (salt concentration) or changing the pH of
elution buffer.

• Ex: protein(s) of interest is eluted by using elution buffer

with various salt concentration (salt gradient) or buffer
with change in pH. – comparison to wash buffer
• Selection of ion exchanger – is determine by the
nature (ion/charge) of molecules

• Selection of buffer (wash, elution) – is determine

by the nature of the ion exchanger and the
nature of biomolecules
- the pH chosen for the buffer depends on the
stability of biomolecules to be separated and
allow binding of molecules to ion exchanger
 Ion exchange chromatography chromatogram

A B C D
A = Equilibration
B = sample binding
Elution
and wash
gradient
curve
(Flowthrough-
unbound/unwanted
Absorption (nm)

molecules)

C= Elution
D=Regeneration
Elution volume or Retention time
 43
From Ion-Exchange Chromatography Principles and Methods
handbook by GE Healthcare
 Ion-Exchange Chromatography
Elution is achieved by
using high ionic
strength salt solution

4. Large net positive charge

3. Net positive charge
2. Net negative charge
Cationic exchanger
1. Large net negative charge

Or Acidic ion
exchanger

44

From Ion-Exchange Chromatography and Its Applications by Özlem Bahadir

Acikara (http://dx.doi.org/10.5772/55744)
 Anion exchange chromatography

Figure 2.12

• negatively charged groups on proteins bind to positively

charged groups on matrix

• increasing salt concentration displaces proteins – protein is

eluted from the column 45
 Affinity Chromatography
• Uses specific binding
properties of
molecules/proteins
• Stationary phase has a
polymer that can be
covalently linked to a
compound called a
ligand that specifically
binds to protein

46
Affinity chromatography
Makes use of specific binding interactions between molecules

1- Incubate crude sample

with the immobilized ligand 3- Elute

47
2- Wash away non bound
sample components from
solid support
Affinity chromatography
• Possible elution strategies:
• pH – change in pH weaken the interaction
• Ion strength – increase buffer ionic strength
• Denature – addition of denaturing agents can
decrease the stability of protein-ligand
• Competitor ligand or analog

48
Group Specific Affinity Resins

49
 4. Protein
CHARACTERIZATION

50

Support materials: Tropp B.E, Molecular Biology Genes to Proteins 3rd Edition, Jones and Bartlett.
Lecture 7
Characterization of separated
proteins
1. Electrophoresis
2. Protein concentration determination –
A280 nm, Lowry assay, Bradford assay
3. Specific assay – enzyme activity
determination, antibody-antigen affinity
binding
51
Electrophoretic technique for
protein
• For protein separation, all methods use
polyacrylamide as sieving matrix
covering a protein size range of 5 to
250 kDa.

• The separation of large proteins or

protein complexes > 300 kDa rely on
the larger pore size of agarose gels.
POLYACRYLAMIDE GEL
ELECTROPHORESIS (PAGE) -
Polyacrylamide Gels
• Polyacrylamide gels are tougher than agarose gels
• Acrylamide monomers polymerize into long chains that are
covalently linked by a crosslinker
• Polyacrylamide is chemically complex, as is the production
and use of the gel

Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)

 PAGE
• This method separates proteins based on their molecular
weights/size and charges---->remember proteins are
zwitterions.

• Each protein component has a unique charge/mass ratio

and discrete size and shape therefore these influence
component mobility

How fast will the protein migrate?

✓ Strength of the electrical field

✓ Charge/mass ratio
✓ Buffer
✓ Concentration of acrylamide gel used
Continuous and Discontinuous electrophoresis
gel systems
• A continuous system has only a single separating gel and uses
the same buffer in the tanks and the gel

• In a discontinuous system a nonrestrictive large pore gel,

called a stacking gel, is layered on top of a separating gel (or
also known as resolving gel)

• The resolution obtainable in a discontinuous system is much

greater than that obtainable in a continuous one. However, the
continuous system is a little easier to set up

Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)

 Continuous and Discontinuous Buffer
Systems

Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)

Discontinuous Polyacrylamide Gel Electrophoresis

pH 6.8

10 – 20 %
acrylamide concentration
pH 8 to 9

The stacking gel has lower ionic strengths and pH also

has lower acrylamide concentration (< 5%)
Direction of protein
migration?
 SDS-PAGE
Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis
• In SDS separations, migration is determined not by intrinsic
electrical charge of polypeptides but by molecular weight

• Sodium dodecyl sulfate (SDS) is an anionic detergent which

denature proteins by binding to the hydrophobic regions thus
denatures secondary and non–disulfide–linked tertiary structures.
In so doing, SDS confers a net negative charge to the polypeptide
in proportion to its length

• When treated with SDS and a reducing agent (eg -

mercaptoethanol), the polypeptides become rods of negative
charges with equal “charge densities" or charge per unit length.

Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)

 SDS-PAGE
• A concurrent treatment with a disulfide reducing agent such
as β-mercaptoethanol or DTT (dithiothreitol), which further
denatures the proteins by reducing disulfide linkages, thus
overcoming some forms of tertiary protein folding, and
breaking up quaternary protein structure

• When treated with SDS and a reducing agent (eg -

mercaptoethanol), the polypeptides become rods of negative
charges with equal “charge densities" or charge per unit
length.
❑ the proteins samples are having uniformed structure and
charge → the separation will depend on their molecular
weight only.
Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)
 SDS-PAGE
How fast will the protein migrate?

✓ Strength of the electrical field

✓ Size of the protein (molecular weight)
✓ Buffer
✓ Concentration of acrylamide gel used
 Native or nondenaturing
PAGE
• Absent of SDS and reducing agent in PAGE
• Separation is based on charge/mass ratio

• Native PAGE is used when the experiment

requires that the protein analyzed retains its
biological activity. ----> example enzyme
experiment
Preparing polyacrylamide gel
• Mix acrylamide stock, TEMED and APS
(last!) in buffer – also SDS for SDS-PAGE
• Pour solution into gel casting plate
• Insert “comb” and let the gel harden.
• Place gel into an electrophoresis tank
• Addition of running buffer
• Load sample into wells
• Attach the tank to powerpack.
A guide to polyacrylamide gel electrophoresis and detection - BioRad
 Protein sample preparation
• Before sample is loaded into well, sample buffer/loading
buffer is added into sample solution.
• Typical loading/sample buffer constituents:
i. dye (tracking dye)
(e.g. bromophenol blue)
ii. High density medium (e.g. glycerol, sucrose)
(reason for having this in sample buffer?)
iii. solvent, generally water
iv. SDS, DTT/-mercaptoethanol – (if SDS-PAGE)

Heat the solution at 70 oC to 100 oC

 Running buffer constituent

• Typical running buffer has Tris, glycine

(nondenaturing). Tris, glycine and SDS are
present in running buffer of the denaturing gel.

• However, other systems that uses MOPS,

MEPS and TRICINE buffer are present.

• pH for PAGE running buffer is between pH 7.0

to pH 9.0 based on pH type.
 During loading of protein samples into wells, protein
standard is added into one well and samples are loaded
in other wells

Staining and detecting PAGE bands

• Tracking dye – For protein electrophoresis,

bromophenol blue is typically used.

• Post-staining is used to stain PAGE.

Analysis of electrophoresis gel
 Protein standard – known as
protein marker/protein ladder
• A standard mixture with known molecular
weights must be added to electrophoresis
under the same condition as samples.

• After electrophoresis and dye staining,

molecular weights are determined
graphically and mobilities are measured.
 Determining Molecular Weights of Proteins

• Run a gel with standard proteins of known

molecular weights along with the polypeptide to
be characterized

• A linear relationship exists between the log10 of

the molecular weight of a polypeptide and its Rf

• Rf = ratio of the distance migrated by the

molecule to that migrated by a marker dye-front

• The Rf of the polypeptide to be characterized is

determined in the same way, and the log10 of its
molecular weight is read directly from the
standard curve

Introduction to electrophoresis (https://ag.arizona.edu/research/ravilab/112408.ppt)

 Electrophoresis
• Electrophoresis- separates molecules according to charge and
size
charged particles migrate in electric field toward opposite
charge
The migration of ions in an electric field to separate molecules.
• Proteins have different mobility:
• Charge
• Size
• Shape

Gel is in vertical 71
orientation
Principle of PAGE REVISION
• Uses acrylamide:bis-acrylamide monomers to form
polyacrylamide gel
• Discontinuos gel – has stacking and resolving gel with different
acrylamide %
• Samples are applied to wells
• Protein migration is in an electric field.
• Migration is from –ve toward +ve charge (cathode to anode)
• Using SDS in sample preparation, all proteins has identical
charge (negatively charged) and shape to mass ratio.
• Separation based on molecular weight; proteins that migrate
fastest have least molecular weight.
• Protein bands are visualized by staining with dyes – eg
72
Coomassie staining
Isoelectric Focusing
Bioseparation based on pI
• Isolectric focusing- based on differing
isoelectric pts. (pI) of proteins
• Gel is prepared with pH gradient that parallel
to the electric field. What does this do?
- Charge on the protein changes as it
migrates.
- When the proteins reach its pI, the
migration stopped because has no charge 73

Gel is in horizontal orientation

IEF: Polyampholytes
Proteins are fed into a gel medium (can be
polyacrylamide, agarose or dextran) that has a
pH gradient to it.

pH gradient is formed by polyampholytes –

added to the gel medium or using a gradient
gel.

Polyampholytes are much like amino acids in

that they are zwitterionic.
74
Principle of IEF
• Definition:-Isoelectric focusing: separation based on
protein pI/charge in an electric field.
• polyacrylamide, agarose or dextran gel treated with
ampholytes; the pH gradient is formed in the gel by
ampholytes
• protein at its isoelectric pH- proteins has no net
charge/no migration/precipitate
• migration from cathode (-ve)(basic pH) and anode
(+ve)(acidic pH) to pI point
• protein bands are visualized by staining with dyes – eg
Coomassie staining
75
 2D-gel electrophoresis

• 2D-gel electrophoresis is an invaluable tool for

proteomics.

• Proteome (like genome) is the total number of all proteins

expressed by a cell or organism, but with an emphasis on
their quantitation, localization, modifications, interactions,
and activities, as well as their identification.

• Individual protein bands from a stained gel can be cut out

of a gel, destained, and the protein can be eluted from the
gel fragment for identification and characterization using
mass spec. 76
 2D-gel electrophoresis

1st running
dimension -
IEF

2nd running
dimension -
PAGE
77

https://kendricklabs.com/2d-overview/
2D-gel electrophoresis
First dimension - IEF
Proteins each have their own isoelectic
point that comes about as an average of
their amino acid isoelectric point.
Proteins are charged at different pHs.
When they reach the region of the gel
that matches their isoelectric point,
they stop moving.
Proteins can be removed at this point
for further analysis if desired.
78
Proteins can also be subjected to
further analysis within the gel.
 2D-gel electrophoresis
Now that the proteins have been separated by isoelectric
point, they can be analyzed based on their mass.
Proteins are separated by mass using SDS-PAGE. SDS acts
as a detergent to uncoil the protein and give it a negative
charge, since the proteins have zero charge after the
isoelectric focusing.

The proteins migrate by applying an

electric field 90 degrees from where it
was located for isoelectric focusing.
Proteins have now migrated in 792
different dimensions from their starting
point, giving a “3D” gel
 VISUALISATION OF PROTEIN BANDS OR SPOTS
IN ELECTROPHORESIS TECHNIQUE

• Now that the proteins have migrated to

their new positions based on isoelectric
point and mass, they can be analyzed.
• Gel can be stained using coomassie or
silver.
• Silver binds to the cysteine groups in
the protein, and leaves a dark stain on
the gel after development.
• Coomassie binds to arginine, histidine,
and aromatic amino acids. It can also
80
be used to replace SDS to give the
negative charge to the protein.
 ANALYSIS OF PROTEIN FROM
2D-ELECTROPHORESIS
• Detection of protein spots using Coomassie and/or Silver
staining.

• Protein spot map analysis using bioinformatic tools –

relate to databases

• Spots are picked and send for protein sequencing – to

confirm the type of protein of the spot

81
The spots can be quantitated, extracted and analysed
Protein sequencing

82

Figure 2.18: Specific cleavage

Edman degradation can be used to
determine protein sequence

Edman degradation – protein is

cleaved into smaller fragments in
stepwise modification starting
from the N-terminal end,
followed by cleavage of each
amino acid in the sequence and
subsequent identification of each
modified amino acid as it is
removed.
PTH-amino acid;
later identified by chromatography
Figure 2.17
Adapted from Tropp, B. E. 83
Biochemistry: Concepts and
Applications. Brooks/Cole
• Edman degradation removes the N-terminal amino acid Publishing Company, 1997.
Amino acid sequence determination

Figure 2.19: The overlap method for amino acid sequence determination.

• Treatment with enzymes or reagents that cleave the protein at specific 84

sites releases small peptides that can be sequenced by Edman
degradation.
• The sequence is constructed by generating overlapping peptide fragments
 PROTEIN SEQUENCING

Mass spectrometry determines

the molecular masses of peptides
Mass spectrometry is a technique for
characterizing and sequencing polypeptides.

By comparing the molecular masses of

successively larger members of a family of
fragments, the identities of the amino acid
residues can be determined – using mass-
comparison via computer programs.

Voet, Voet, and Pratt (2008). Chapter 5 page 110

Figure 2.20 85
Adapted from Steen, H., and Mann, M., Nature Rev. Mol. Cell Biol. 5 (2004): 699-711.
 HIGH-THROUGHPUT PROTEIN
X-ray crystallography to CHARACTERIZATION TECHNIQUE

determine protein structure

BMS535 Protein & Enzymes

86
Figure 2.34
Adapted from Branden, C. I., and Tooze, J. Introduction to
Protein Structure, Second Edition. Routledge, 1999.
 X-ray crystallography
• A technique that directly images molecules
• Protein is crystallized and subjected to X-rays (generate by
synchrotrons; particle accelerators that produce X-rays of
greater intensity) – resulting in diffraction pattern
• X-rays interact with electrons in protein crystal thus produces
an image of the electron density of the crystal.
• Via electron density map, the atomic positions of the amino
acids in the protein are interpreted.
Voet, Voet, and Pratt (2008). Chapter 6 page 141

87

Hampton Research crystal gallery Diffraction pattern Electron density map

NMR spectroscopy can be used to determine the
structure of small proteins HIGH-THROUGHPUT PROTEIN
CHARACTERIZATION TECHNIQUE

BMS535 Protein & Enzymes

88
Adapted from Berg, J. M., et al. Biochemistry, Fifth Adapted from Branden, C. I., and Tooze, J. Introduction to
Edition. W. H. Freeman and Company, 2002. Figures 2.36 and 2.37 Protein Structure, Second Edition. Routledge, 1999.
 Nuclear Magnetic Resonance (NMR)
• A technique that observe an atomic nucleus such as a proton
(a hydrogen nucleus) resonates in an applied magnetic field.
• The chemical shifts of atomic nucleus is determine and a
spectrum of atoms are form, each peak correlate to amino
acid residues.
• Interatomic distance measurements, prior knowledge of
protein sequence, covalent bond distances, angles, van der
Waal radii are used to compute the protein’s three
dimensional structure. Voet, Voet, and Pratt (2008). Chapter 6 page 143

89

NMR spectra
 Summary of techniques use in
protein purification
• Tissue/cell lysis techniques - osmolysis, mechanical disruption-high
speed blender, homogenizer, French press, sonication
• Protein precipitation using Salting out and salting in
• Protein separation using chromatography
• Ion exchange
• Size exclusion
• Affinity
• Dialysis – separate small solutes from protein mix, also can be used
to buffer exchange protein solution
• Protein characterization and identification using electrophoresis
• SDS PAGE
• 2D electrophoresis
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• Protein characterization
• Isoelectric focusing, mass spectrophotometry
 Summary of techniques use in
protein characterization
• PAGE (Polyacrylamide gel electrophoresis) as example 1D/2D
electrophoresis –identify protein molecular weight
• IEF (Isoelectric focusing) - characterize protein pI
• Size exclusion chromatography –identify protein molecular weight
• ELISA (Enzyme-linked immunoabsorbent assay) – to characterize
antibody-antigen interaction
• SPR (Surface Plasmon Resonance) – characterize protein-protein
interaction example antibody-antigen association and protein-
receptor interaction.
• Mass spectroscopy – identify molecular weight of protein and also
determination of amino acid in protein. 91
• X-ray crystallography and NMR – determination of protein structure.