Lesson

Antibody
Production

The blood contains two types of white blood

cell or leukocyte
Phagocytes ingest bacteria by endocytosis

Lymphocytes produce antibodies

Antibodies
Antibodies are proteins that recognise and
bind to specific antigens
Antigens are foreign substances that
stimulate the production of antibodies
Many of the molecules on the surface of
viruses and bacteria are antigens

Antibodies are specific they usually bind

to only one specific antigen.
Antibodies

Antigen

Microbe

Production of Antibodies by Lymphocytes

A lymphocyte can produce only one type of
antibody so a huge number of different types are
needed
Each lymphocyte has some of its antibody on its
surface

The antigens of a pathogen bind to the antibodies

in the surface membrane of a lymphocyte

This activates the lymphocyte.

The active lymphocyte divides by mitosis to

produce a clone of many identical cells
MITOSIS

The clone of cells

starts to produce large
quantities of the same
antibody
the same antibody
needed to defend
against the pathogen!

Most microbes have more

than one antigen on their
surface, so
they stimulate more than
one type of lymphocyte
resulting in the production
of many different antibodies.
These are called
polyclonal antibodies.

Antibody Production: The Primary Response

Step 1: Antigen Presentation
Antigen

Macrophage

Macrophages take in antigen

by endocytosis
The macrophage processes the
antigen and attaches it to a
membrane protein called a
MHC protein

The MHC protein is moved to the

cell surface membrane by
exocytosis so that the antigen is
displayed on its surface.

MHC protein

Step 2: Activation of Helper T-cell

Helper T-cells have receptors on
their cell surface membranes which
can bind to antigens presented by
macrophages.

receptor

Helper T-cell

Helper T-cell binds to macrophage

presenting the antigen

Macrophage sends a signal to activate the helper T-cell

Step 3: Activation of B-lymphocytes

B-cells have antibodies in their cell surface
membranes
Antigens bind to the antibodies in the surface
membranes of B-cells
Antigen
Inactive B-cell

Antibody

An activated helper T-cell with

receptors for the same antigen
binds to the B-cell

SIGNAL

The helper T-cell sends a signal to the B-cell,

activating the B-cell.

Step 4: Proliferation
The activated B-cell starts to divide
by mitosis to form a clone of plasma
cells.
Plasma cells are activated B-cells
with a very extensive network of
rough endoplasmic reticulum.
Plasma cells synthesis large
amounts of antibody, which they
excrete by exocytosis.

The Secondary Response: Memory Cells

If an antigen invades your body a second time, a
much faster response occurs which produces
much larger quantity of the required antibody.
When activated B-cells are dividing during the
primary response, some cells stop dividing and
secreting antibody and become memory cells.
Large numbers of memory cells remain in the
body for a long time
they are capable of producing large amounts of
antibody very quickly when stimulated.

Antigen

Antigen

Activate

B-cell
Clone

Memory
Cell

Activate

Helper
T-cell

Antibody Production:
Summary

Plasma
Cell

Macrophage

Antibodies

Vaccine Development
Traditional and Modern
Approaches

Purpose of Vaccina6on

18

Purpose of Vaccina6on

Protect the individual from disease.

Reduce the severity of disease.
Protect the community.
Eradica6on of the disease.

The value of Vaccines

Vaccines are the most cost-effective
tools for preventing death and
disability from infectious disease
Luis Fermn Tenorio,
the last polio case in
the Americas
Peru, 1991
Ali Maouw Maalin,
last case of smallpox
(Somalia,1977)
20

Normal Bacterial Flora

Staphylococcus epidermidis

Normal
colon

Bacterial colonization of
the body
per gm or cm2

Bacterial

Bifidobacterium bifidum
E.coli

Bacterial Toxins in disease

DISEASE
(BACTERIA)
Diphtheria
Corynebacterium diphtheriae

Tetanus
Clostridium tetani

Whooping cough
Bordetella pertussis

Cholera
Vibrio cholerae

TOXIN PRODUCED
Diphtheria toxin
Tetanus toxin
Adenylate cyclase toxin
Pertussis toxin
Cholera enterotoxin

Invasion of Cells

Shigella bacterium invading HELA cell

Basic concept of vaccines

Deliver to the body some part or all of the disease
organism that IMITATES the pathogen but is not
pathogenic.
Induce protec1ve immune response.

Polysaccharide
LP
S capsu
Intracellular
lar
proteins
Entire
Surface
organism
proteins
live
Toxins
(attenuated)
killed

Vaccine manufacture
Antigen Production

Eggs

Bacterial / Yeast fermenta1on

Inuenza

Whole organism (e.g. Cholera)

Subunit vaccines (e.g. Capsular polysaccharide,
Tetanus and Diphtheria toxoid)
Gene1cally engineered proteins (e.g. Hepa11s B
and HPV vaccines)

Cell culture

Viral vaccines either whole virus or subunit

Gene1cally engineered proteins
25

Vaccine Processing

An6gen concentra6on.
Removal of unwanted foreign components.

Host proteins
Host DNA
Adven66ous agents

Removal of unnecessary Bacterial or Viral

components.

LPS
Unwanted proteins (not involved in immunity)
Unwanted nucleic acid

Change of dilu6ng solu6on.

Addi6on of other components.

Adjuvants, stabilizers, preserva6ves etc

Growth
of
Virus, Yeast
or Bacteria

Eggs

Clarification

Concentra1on

Purica1on

Depth lter
Crossow
ltra1on

Tools available to develop a process

Chromatography
Ion exchange

Bacterial / Yeast

Crossow ltra1on

Hydrophobic
Anity

Fermenta1on
Ultracentrifuga1on

Cell culture

Centrifuge

Size exclusion

Chemical
Formaldehyde

precipita1on

Inactivation
PL

Antigen
presentation

DT

Formulation

CONHNHCO(CH2)4CONHNHOC-

Vi-CPS

CONJUGATES

Combining antigen(s)
Combining with
adjuvant
Stabilizers

VIRUS LIKE PARTICLES

ISCOMS

Preservatives
Cryo-protectants

ADJUVANTS

VIROSOMES

Fill and Finish

Types of Vaccines

29

Types of Vaccines

Inac6vated toxins
Inac6vated whole bacteria or viruses
Live aRenuated bacteria or viruses
Subunit vaccines
Gene6cally engineered proteins
Polysaccharide vaccines
Conjugated vaccines
Recombinant DNA modied organisms
DNA vaccines
30

Inac6vated Toxins
Exotoxin
Gram nega6ve and gram
posi6ve bacteria.
Heat Labile.
Protein.
Secreted by the bacteria.

Endotoxin
Gram nega6ve bacteria.
Heat stable.
Lipopolysaccharide.
Firmly bound to the
bacteria outer membrane.

31

Toxoid
Not a vaccine against the organism.
Vaccine against pathogenic exotoxin.
Tetanus, diphtheria, (pertussis?), anthrax?
Purify toxin then chemically inac6vate (toxoid)
Risk of incomplete inac6va6on.
TT, DT.

Gene6cally modify toxin so non-toxic

CRM (diphtheria), mLT (cholera, ETEC)
32

Inactivated Toxins

Tetanus toxin blocks

neurotransmission
from nerve endings
inducing muscle

Diphtheria toxin
inhibits protein
synthesis resulting in
cell death

33

Tetanus

34

Tetanus

Clostridium tetani
Gram posi6ve drum s6ck shaped rod
Not transmiRed from person to person
Transmission via contaminated wounds
Found in soil and animal feces
Replicate in low oxygen environment
Release toxin which enters bloodstream
Acts on the nervous system to block
neurotransmiRers
35

Tetanus
The toxin causes the disease.
The vaccine consists of toxin which is rendered inac6ve
(toxoid) but retains immunogenicity.
The immune response to the vaccine is directed against the
toxin rather than the bacteria.
Vaccina6on results in serum an6body produc6on. The
an6body neutralizes toxin.
Three doses of vaccine recommended to raise an6body levels
to protec6ve levels.
Immunity lasts about 10 years.
Vaccina6on prevents disease but is incapable of disease
eradica6on.
36

Neonatal and Maternal Tetanus

Tetanus in Children has been largely controlled by
vaccina6on.
In 1999, WHO es6mated 290,000 cases of
neonatal tetanus which resulted in 14% (215,000)
of neonatal deaths. 30,000 (5%) of Maternal
deaths were due to tetanus.
Target to eliminate neonatal and maternal deaths
due to tetanus by 2005 by vaccina6on of women
of child bearing age.
37

Diphtheria

38

Diphtheria
Corynebacterium diphtheriae.
Gram posi6ve bacillus.
Only produces a powerful exotoxin when infected
with a bacteriophage. (the phage carries the toxin
gene which coverts the bacteria from non-toxigenic
to toxigenic)
Diphtheria is now well controlled by na6onal
immuniza6on programs.
Only receives aRen6on when rou6ne immuniza6on
programs fall.
39

Diphtheria

40

Recombinant
Advantage
Safe. Growth in non pathogenic yeast cells
Easier in case of dicult to grow viruses like
hepB, HPV.

Disadvantages:
Need to iden6fy protec6ve an6gen/s
Obtaining an6gen in 'correct' conforma6on
Usually poorly immunogenic alone
Poor CMI requires adjuvant.
41

Recombinant Vaccines

E.coli

S. cerevisiae

42

Recombinant Vaccine

43

Recombinant Vaccine
The gene coding for HBsAg was discovered in
1970.
The gene has been inserted into a yeast cell.
As the yeast cell grows it produces large
amounts of HBsAg.
The HBsAg is extracted and puried then
incorporated into the vaccine.

44

Recombinant Vaccine
Advantages of the recombinant vaccines.
Produced more quickly.
In larger quan66es.
Free from infec6ous virus par6cles.

45

Recombinant DNA modied organisms

Live Vectors

Cloning of gene6c material from one organism

into another.
The non virulent parent organism expresses
the an6gens of the cloned gene6c material.
A vaccine would elicit a response against the
introduced an6gen as well as the original
organism.
46

Recombinant DNA modied organisms

Vaccinia virus
expressing papilloma
virus antigens on its
surface.

47

DNA Vaccines
Involves the injec6on of naked DNA coding for
one or more genes.
The gene is grahed onto another piece of DNA
which acts as a vector.
Injected into muscle 6ssue, once in the cell
the gene prompts the cell to produce an6gen.
The immune system then mounts an immune
response.
48

DNA Vaccines
Viral
protein

mRNA

Antibodyproducing cell

Nucle
us
Injected
DNA coding

for a specific
Class1
antigen
MHC
Cytotoxic Tlymphocyte

49

DNA Vaccines
Clinical trials to date with naked DNA vaccines
have not proved to be that successful
DNA vaccines may be useful as a priming dose
in prime-boost regimes due to their ability to
induce cell mediated immune responses.

50

Protein Purica6on
The aim of protein purica6on is to isolate
one par6cular protein from all the others in
the star6ng material.
A combina6on of frac6ona6on techniques is
used that exploits the solubility, size, charge
or/and specic binding anity of the protein
of interest.

Selec6on of Protein Source

Because proteins have dierent distribu6ons
in biological materials, it is important to make
the right choice of star6ng material from
which to purify the protein.
This will usually be a source that is rela6vely
rich in the protein of interest and which is
readily available.

Homogeniza6on and Solubiliza6on

The protein has to be obtained in solu6on prior
to its purica6on.
Thus 6ssues and cells must be disrupted by
homogeniza6on or osmo6c lysis and then
subjected to dieren6al centrifuga6on to isolate
the subcellular frac6on in which the protein is
located.
For membrane-bound proteins, the membrane
structure has to be solubilized with a detergent
to liberate the protein.

Stabiliza6on of Protein
Certain precau6ons have to be taken in order
to prevent proteins being denatured or
inac6vated during purica6on by physical or
biological factors.
These include buering the pH of the
solu6ons, undertaking the procedures at a low
temperature and including protease inhibitors
to prevent unwanted proteolysis.

Assay of Protein
In order to monitor the progress of the
purica6on of a protein it is necessary to have
an assay for it.
Depending on the protein, the assay may
involve measuring the enzyme ac6vity or
ligand-binding proper6es, or may quan6fy the
protein present using an6bodies directed
against it.

Ammonium Sulfate Precipita6on

The solubility of proteins decreases as the
concentra6on of ammonium sulfate in the
solu6on is increased.
The concentra6on of ammonium sulfate at
which a par6cular protein comes out of
solu6on and precipitates may be suciently
dierent from other proteins in the mixture to
eect a separa6on.

8
9
30111
1
Dialysis
2
3
4
5
6
7
8
9
40111
1
2
3
4
5
6
7
8
9
50
5111

Dialysis
Proteins can be separated
from small molecules by
dialysis through a semi-
permeable membrane
which has pores that allow
small molecules to pass
through but not proteins.

cation procedure to concentrate a dilute solution of the prote

precipitates and can then be redissolved in a smaller volum

Proteins can be separated from small molecules by dialys

permeable membrane such as cellophane (cellulose ace
membrane allow molecules up to approximately 10 kDa
whereas larger molecules are retained inside the dialysis ba
proteins have molecular masses greater than 10 kDa, this te
able for fractionating proteins, but is often used to remove sm
(a)

(b)

Fig. 1. Separation of molecules on the basis of size by dialysis. (a) St

(b) at equilibrium.

Ultracentrifuga6on
Proteins with large
dierences in
molecular mass can
be separated by
ratezonal
centrifuga6on using
a gradient of a
dense material such
as sucrose.

(A)

armored
chamber

sedimenting
material

(B)

sedimenting
material

hinge

cell
homogenate
rotor

LOW-SPEED CENTRIFUGATION

refrigeration

pellet contains
whole cells
nuclei
cytoskeletons
SUPERNATANT SUBJECTED TO
MEDIUM-SPEED CENTRIFUGATION

pellet contains
mitochondria
lysosomes
peroxisomes
SUPERNATANT SUBJECTED TO
HIGH-SPEED CENTRIFUGATION

pellet contains
microsomes
small vesicles
SUPERNATANT SUBJECTED TO
VERY-HIGH-SPEED CENTRIFUGATION

pellet contains
ribosomes
viruses
large
macromolecules

motor

vacuum

refrigeration

motor

vacuum

Figure 85 The preparative ultracentrifuge. (A) The sample is contained in tubes that are

inserted
into a ring of angled cylindrical holes in a metal rotor. Rapid rotation of the rotor generates
PURIFYING
PROTEINS
enormous centrifugal forces, which cause particles in the sample to sediment against the bottom
sides of the sample tubes, as shown here. The vacuum reduces friction, preventing heating of the
rotor and allowing the refrigeration system to maintain the sample at 4C. (B) Some fractionation
(A)
(B)
methods require a different type of rotor called a swinging-bucket rotor. In this case, the sample
EQUILIBRIUM
VELOCITY
MBoC6
tubes
are placed in metal tubes on hinges
that m8.09/8.07
allow the tubes to swing
outward when the rotor
SEDIMENTATION
SEDIMENTATION
spins. Sample tubes are therefore horizontal during spinning, and samples are sedimented toward
the bottom, not the sides, of the tube, providing better separation of differently sized components
sample
(see Figures 86 and
87).

sample
stabilizing shallow
sucrose
LOW-SPEED CEN
gradient
steep
(e.g.,
520%)
sucrose
Centrifugation
is the
first step in most fractionations, but it separates only
comgradient
ponents that differ greatly in size. A finer degree of separation can be achieved
(e.g., by
2070%)

layering the homogenate in a thin band on top of a salt solution that fills a centricentrifuged, the various components in the mixture move as a
series of distinct bands through the solution, each at a different rate, in a process
called velocity sedimentation (Figure 87A). For the procedure to work effectively,
the bands mustslow-sedimenting
be protected from convective mixing, which would normally
SUPERNATANT S
occur whenevercomponent
a denser solution (for example, one containing organelles) finds
MEDIUM-SPEED
itself on top of afast-sedimenting
lighter one (the salt solution). This is achieved by augmenting
component
the solution in the
tube with a shallow gradient of sucrose prepared by a special
mixing device. The resulting density gradientwith the dense end at the bottom
of the tubekeeps each region of the solution denser than any solution above it,
FRACTIONATION
and it thereby prevents convective mixing from distorting the separation.
When sedimented through sucrose gradients, different cell components sepalow-buoyantrate into distinct bands that can be collected individually. The relative rate at
which
density
each component sediments depends primarily on its size and shapenormally
component SUPERNATANT S
HIGH-SPEED CEN
being described in terms of its sedimentation coefficient, or S value. Present-day
high-buoyantultracentrifuges rotate at speeds of up to 80,000 rpm and produce forces asdensity
high as
component
500,000 times gravity. These enormous forces drive even small macromolecules,
such as tRNA molecules and simple enzymes, to sediment at an appreciable rate
and allow them to be separated from one another by size.
The ultracentrifuge is also used to separate cell components on the basis of
their buoyant density, independently of their size and shape. In this case, the

CENTRIFUGATION
fuge tube. When

SUPERNATANT S

VERY-HIGH-SPEE
Figure 86 Cell fractionation by centrifugation. Repeated centrifugation
at progressively higher speeds will fractionate homogenates of cells into their
sample is sedimented through
a general,
steepthedensity
gradient
that contains
a very high
components. In
smaller the subcellular
component,
the greater
concentration of sucrose
cesium
chloride.
Each
cellvalues
component
the or
centrifugal
force required
to sediment
it. Typical
for the variousbegins to move

1111
2
3
4
5
6
7
8
9
10111
1
2
3
4
5

Gel Filtra6on Chromatography

Gel ltra6on chromatography separates proteins on the basis of their size
and shape using porous beads packed in a column.
Large or elongated proteins cannot enter the pores in the beads and elute
from the boRom of the column rst, whereas smaller proteins can enter
the beads, have a larger volume of liquid accessible to them and move
through the column more slowly, elu6ng later.
B7
Gel ltra6on chromatography can be used to desalt a protein mixture and
Chromatography of proteins
to es6mate the molecular mass of a protein.
(a)

Porous
beads

Mixture of
proteins

Buffer added to top

Large
molecules

Small
molecules

Glass/
plastic
column
Tube to
collect
protein

Large

Small

sample
applied

solvent continuously
applied to the top of
column from a large
reservoir of solvent

solid
matrix
porous
plug
test
tube
time

fractionated molecules
eluted and collected

Figure 88
by column
a solution c
molecules,
cylindrical g
a permeable
A large amo
slowly throu
in separate
bottom. Bec
the sample
the column,
different tub

Ion Exchange Chromatography

In ion exchange chromatography, proteins are separated on the basis of
their net charge.
In anion exchange chromatography a column containing posi6vely
charged beads is used to which proteins with a net nega6ve charge will
bind, whereas in ca6on exchange chromatography nega6vely charged
beads are used to which proteins with a net posi6ve charge will bind.
The bound proteins are then eluted by adding a solu6on of sodium
chloride or by altering the pH of the buer.
Section B Amino acids and proteins

+
Positively
charged
beads

+
+
+

Glass
column

Buffer

Mixture of
proteins

(a)

+
+

+
+
+
+

NaCl

+
+
+

Positively
charged
proteins

Negatively
charged
proteins

+
+

+ Cl
+
Cl
+
+

Cl
+

+
Cl
+
+
+
+
+
+
+

+
+
+
+

Tubes to
collect
protein

Negatively
charged
proteins

Anity Chromatography

Anity chromatography exploits the specic binding of a protein

for another molecule, its ligand (e.g. an enzyme for its inhibitor).
The ligand is immobilized on an insoluble support and packed in a
column. On adding a mixture of proteins, only the protein of
interest binds to the ligand.
All other proteins pass straight through.
The bound protein is then eluted from the immobilized ligand in a
B7 Chromatography of proteins
highly puried form.
(a)

Ligand
immobilized
on insoluble
support and
packed in
glass column

Mixture of
different
proteins

Specific
protein
binds to
ligand

Unwanted
proteins pass
straight through

Soluble ligand
added

Specific
protein elutes
off bound to
soluble ligand

PURIFYING PROTEINS
solvent flow

+ ++
positively
+
+
charged
+
+
+
+
bead
+ +
+ +
+
+ +
+
+
+
+ bound
+
+++
negatively
+ +++
+ + charged
+ +
molecule
+
+
+
+
+
free
+++ +
+
positively
+ +
+
charged
molecule

449
solvent flow

solvent flow

(A) ION-EXCHANGE CHROMATOGRAPHY

porous bead

retarded
small molecule
unretarded
large molecule

(B) GEL-FILTRATION CHROMATOGRAPHY

a means of separating molecules, gel-filtration chromatography is a convenient

bead with
covalently
attached
substrate
bound
enzyme
molecule
other proteins

(C) AFFINITY CHROMATOGRAPHY

Figure 89 Three types of matrices

Na6ve PAGE
In na6ve polyacrylamide
gel electrophoresis (PAGE)
proteins are applied to a
porous polyacrylamide gel
and separated in an electric
eld on the basis of their
net nega6ve charge and
their size.
Small/more nega6vely
charged proteins migrate
further through the gel
than larger/less nega6vely
charged proteins.

SDS-PAGE
In SDS-PAGE, the protein sample is treated with a reducing
agent to break disulde bonds and then with the anionic
detergent sodium dodecyl sulfate (SDS) which denatures
the proteins and covers them with an overall nega6ve
charge.
The sample is then frac6onated by electrophoresis through
a polyacrylamide gel.
As all the proteins now have an iden6cal charge to mass
ra6o, they are separated on the basis of their mass.
The smallest proteins move farthest.
SDS-PAGE can be used to determine the degree of purity of
a protein sample, the molecular mass of a protein and the
number of polypep6de subunits in a protein.

ded onto gel

ipette(A)
cathode

(B)
sample loaded onto gel
by pipette

plastic casing

plastic casing

buffer
gel

buffer

+ anode

+ anode

protein with two

(B)
protein
with two
subunits,
A and
B,
subunits,
A and B,
joined by
a disulfide
joined by a disulfide
bridge
bridge
A
B
A
B
S-S
S-S

single-subunit
single-subunit
protein
protein
C
C

HEATED WITH SDS AND MERCAPTOETHANOL

HEATED WITH SDS AND MERCAPTOETHANOL
_
_
_ __
__ _
_ ___
_
__ __ __ ___ __________
_
_
__ __ _
__ _
_
_
_ _____ __ __
___ __
_
_
_
_
_
_
_ _ __
_
__ _ _
__ ___
_
_ __
_ _ ___
__
__
_
_
___ _ _ _ __ _ _ _ _ __
_
_
_
_
_
_ ___ _ _
_
_
_
_
__ _ _ ___
_ _ _ ___ _ _ __SH
_
_
_
_
_
_
_
_
_ ___ _ _ __
__ __
_
_
_
_
_
_
_
_
SH
_
_
_
_
_
_
__
_ _ _ _ __
_
_
____ _ _
___
_ __ ___ ___ _
__ ___ _
__
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
HS
_
_
_
_
_
_
_
_
_
_
_
_
_
HS
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_
_ _ _ _ ____ ___ negatively
___ _
_
_ _ _ _ __
_
_
__ _
_ negatively
__ __ ____ ___ __ _
__ ___ _
___ ____
_ _ _ _____ _ ___ __ _ _ ____
charged
SDS
C
SDS
C
_ ___
__ charged
_ _____
molecules
_ ___ __ molecules
_
A
A
BB
POLYACRYLAMIDE-GEL
ELECTROPHORESIS
POLYACRYLAMIDE-GEL ELECTROPHORESIS

Figure
813 SDS polyacrylamide-gel
electrophoresis(A)
(SDS-PAGE).
(A) An
de-gel
electrophoresis
(SDS-PAGE).
An
B
electrophoresis
apparatus.chains
(B) Individual
polypeptide
chains form a complex
ndividual
polypeptide
form
a complex
negativelydodecyl
charged molecules
of sodium
dodecyl
sulfate (SDS) and
es ofwith
sodium
sulfate
(SDS)
and
C
therefore
migrate
as
a
negatively
charged
SDSprotein
complex
through a
y charged SDSprotein complex through
a
porous
gel speed
of polyacrylamide.
Because theunder
speed ofthese
migration under these
ecause
the
of migration
conditions
is
greater
the
smaller
the
polypeptide,
this
the polypeptide, this technique can betechnique
used can be used
to
determine
the
approximate
molecular
weight
of
a
polypeptide
chain as
molecular weight of a polypeptide chain as
A
well
as
the
subunit
composition
of
a
protein.
If
the
protein
contains
a large
of a protein. If the protein contains a large
of carbohydrate,
however, it will
the gel and its
er, it amount
will move
anomalously
onmove
theanomalously
gel andon
its
apparent molecular weight estimated by SDS-PAGE will be misleading. Other
mated by SDS-PAGE will be misleading. Other
modifications, such as phosphorylation, can also cause small changes in a
rylation, can also cause small changes in a
proteins migration in the gel.

B
C

+
slab of polyacrylamide gel
slab of polyacrylamide gel

Isoelectric Focusing
In isoelectric focusing,
proteins are separated by
electrophoresis in a pH
gradient in a gel.
They separate on the basis of
their rela6ve content of
posi6vely and nega6vely
charged residues.
Each protein migrates
through the gel un6l it
reaches the point where it
has no net charge, its
isoelectric point (pI).

Visualiza6on of Proteins in Gels

Proteins can be visualized
directly in gels by staining
them with the dye
Coomassie brilliant blue or
with a silver stain.
Radioac6vely labeled
proteins can be detected
by overlaying the gel with
X-ray lm and observing
the darkened areas on the
developed autoradiograph
that correspond to the
radiolabeled proteins.

Western Blot
A specic protein of
interest can be
detected by
immunoblot (Western
blot) following its
transfer from the gel to
nitrocellulose using an
an6body that
specically recognizes
it.
This primary an6body is
then detected with
either a radiolabeled or
enzyme-linked
secondary an6body.

Amino Acid Composi6on Analysis

The number of each type of amino acid in a
protein can be determined by acid hydrolysis
and separa6on of the individual amino acids
by ion exchange chromatography.
The amino acids are detected by colorimetric
reac6on with, for example, ninhydrin or
uorescamine.

Edman Degrada6on
The N-terminal amino acid of a protein can be
determined by reac6ng the protein with dansyl
chloride or uorodinitrobenzene prior to acid
hydrolysis.
The amino acid sequence of a protein can be
determined by Edman degrada6on which sequen6ally
removes one residue at a 6me from the N terminus.
This uses phenyl isothiocyanate to label the N-terminal
amino acid prior to its release from the protein as a
cyclic phenylthiohydantoin amino acid.

Edman Degrada6on
In order to sequence an en6re protein, the polypep6de chain has to
be broken down into smaller fragments using either chemicals (e.g.
cyanogen bromide) or enzymes (e.g. chymotrypsin and trypsin).
The resul6ng smaller fragments are then sequenced by Edman
degrada6on.
The complete sequence is assembled by analyzing overlapping
fragments generated by cleaving the polypep6de with dierent
reagents.
Aminopep6dase and carboxypep6dase release the N- and Cterminal amino acids from a protein, respec6vely.
The polypep6des in a mul6-subunit protein have to be dissociated
and separated prior to sequencing using urea or guanidine
hydrochloride which disrupt noncovalent interac6ons, and 2mercaptoethanol or dithiothreitol that break disulde bonds.

Protein Sequencing By Mass Spec

Short polypep6des can be sequenced rapidly
by fast atom bombardment mass
spectrometry (FAB-MS).
This technique not only provides the amino
acid sequence of the pep6de but also
informa6on on post-transla6onal
modica6ons.

Chapter 8: Analyzing Cells, Molecules, and Systems

(A) STANDARD MASS SPECTROMETRY (MS)

peptide
mixture

+
+

+
+

ion source

+ +
+

+
+

+ +

mass
analyzer

+
+

relative abundance

100

detector

1000

(B) TANDEM MASS SPECTROMETRY (MS/MS)

inert gas

+
+

+
+

ion source

+
+
+

MS2

+
+

2500

100

peptide
mixture
MS1

1500
2000
mass-to-charge ratio (m/z)

mass fragmentation mass

detector
filter
analyzer
(precursor ion
(product ion
selection)
analysis)

relative abundance

456

200

600
mass-to-charge ratio (m/z)

1000

Knowing is not enough; we must apply.

Willing is not enough; we must do.
-Goethe