Core Course # MM 707
(Techniques in Molecular Medicine-II)
Biochemistry
Electrophoresis
Western Blotting
1
Electrophoresis
• Introduction
• Theory and Basic Concepts
• Types of Electrophoresis
SDS-PAGE electrophoresis
Native gel electrophoresis
• Trouble shooting
Introduction
3
Electrophoresis:
 The transport of particles through a solvent by an electric field is
called electrophoresis.
 In the biological system, many molecules are electrically charged
and will move if electric field is applied.
 In electrophoresis, macromolecules are
characterized by their rate of movement in an
electric field.
This technique is used to (1) distinguish
molecules on the basis of charge and shape (2)
to determine molecular weight of proteins (3) to
detect amino acid changes from charged to
uncharged residues & (4) to separate different
molecular species quantitatively.
4
Theory & Basic Concepts
5
What is happening during Electrophoresis: Some
Basic Concepts
• Separation of large (macro) molecules depends upon two forces:
charge and mass.
• When a biological sample, such as proteins or DNA, is mixed in a
buffer solution and applied to a gel, these two forces act together.
• The electrical current from one electrode repels the molecules while
the other electrode simultaneously attracts the molecules.
• The gel material acts as a "molecular sieve," separating the
molecules by size. Molecules are forced to move through the pores
when the electrical current is applied.
6
The rate of migration through the electric field depends on the
(1) strength of the field
(2) size and shape of the molecules,
(3) on the ionic strength and temperature of the buffer in which the
molecules are moving.
7
There is also a frictional resistance that slows down the
movement of this charged molecule. This frictional force is
a measure of the
(1) hydrodynamic size of the molecule
(2) the shape of the molecule
(3) the pore size of the medium &
(4) viscosity of the buffer
8
Mobility depends on charge
Mobility depends on frictional coefficient which in turn
depends on physical parameters of molecules
Therefore, “mobility” gives information about the charge,
size and shape of the molecule
9
• The current in the solution between the electrodes is conducted
mainly by the buffer ions with a small proportion being conducted
by the sample ions.
• It is possible to accelerate an electrophoretic separation by
increasing the applied voltage, which would result in a
corresponding increase in the current flow.
• The distance migrated will be proportional to current
• However, increasing the voltage would result in the generation of
heat.
10
Heating of the electrophoretic medium can have the following
effects:
1. An increased rate of diffusion of sample and buffer ions
leading to broadening of the separated samples.
2. Thermal instability of samples that are rather sensitive to heat.
This may include denaturation of proteins or loss of activity of
enzymes.
3. A decrease of buffer viscosity, and hence a reduction in the
resistance of the medium.
11
Types of Electrophoresis
12
Types of Electrophoresis
Moving Boundary
Zone
Paper
Gel
Polyacryalmide
Non Dissociating
(Native-PAGE)
Agarose
Dissociating
(SDS-PAGE)
13
Gel electrophoresis
* It is used for the separation of proteins and nucleic acids
* Many types of gels are used as supporting medium e.g. starch,
polyacrylamide and agarose
* Earliest work in gel electrophoresis was done with starch
* It provided the first evidence for the existence of isozymes.
* Generally polyacrylamide gels are used for proteins and agarose for
nucleic acids
14
Polyacrylamide Gel
Electrophoresis
15
Polyacrylamide Gel Electrophoresis (PAGE)
Gel Ingredients
1. Acrylamide
2. Bis acrylamide
3. Tris-HCl
4. N,N,N’,N’-tetramethylethelenediamine (TEMED)
5. Ammoinium per sulfate (APS)
16
Acrylamide, Bis Acrylamide & Polyacrylamide
17
Pore size
1.
Pore size of polyacrylamide gel is dependent on the
concentration of acrylamide
2.
Pore size decreases as the conc. of acryalmide increases
3.
High concentration of gels would be able to separate low
molecular weight proteins
18
APS & TEMED
Chemical and Photochemical
•
Polymerization is initiated by APS or riboflavin
•
Chemical: In TEMED-APS system, free base of TEMED
catalyzes the formation of free radicals from persulphate and
these in turn initiate polymerization. TEMED acts as the
accelerator to polymerization process
•
Photochemical: Light is required for initiation of
polymerization. Light causes photodecomposition of
riboflavin and produce free radicals.
•
Oxygen inhibits polymerization
19
20
SDS-PAGE (Dissociating Buffer System)
(Laemmli, 1970)

The buffer system tends to dissociate all proteins into their
individual polypeptide subunits.

Most common dissociating agent is sodium dodecyl suphate,
an anionic detergent.

Before electrophoresis, samples are treated with a solution
containing b-mercaptoethanol and SDS and heated at 1000C.

b-mercaptoethanol is a reducing agent that cleaves disulfide
bonds.

SDS denatures proteins and coats proteins with negative
charges, which overwhelm the protein’s intrinsic charge. Most
polypeptides bind SDS at a constant ratio (i.e. 1.4 g per gram
21
polypeptide)
 Thus, treated proteins assume a rod-like shape and all carry
the same net negative charge. When treated proteins are
electrophoresed through a SDS-PAGE gel, proteins
migrate toward the positive electrode. The proteins can
now be separated by their molecular weights.
22
(a)
(b)
(c)
23
Native-PAGE (Non Dissociating Buffer System)
• "Native" or "non-denaturing" gel electrophoresis is run in the absence of SDS.
Since the protein retains its folded conformation, the mobility depends on both
the protein's charge and its hydrodynamic size.
• The electric charge driving the electrophoresis is governed by the intrinsic
charge on the protein at the pH of the running buffer. This charge will, of
course, depend on the amino acid composition of the protein as well as posttranslational modifications.
• The higher mobility is for more compact conformations, & lower for larger
structures. If native PAGE is carried out near neutral pH to avoid acid or
alkaline denaturation, then it can be used to study conformation, selfassociation or aggregation, and the binding of other proteins or compounds.
• Another advantage of native gels is that it is possible to recover proteins in
their native state after the separation. Recovery of active biological materials
may, however, need to be done prior to any fixing or staining
24
Continuous and Discontinuous Buffer Systems
Continuous:
 Same buffer ions are present throughout the sample, gel and electrode
reservoirs
 Sample is loaded directly on to the gel in which the separation will
occur, called the separating or resolving gel which has pores
sufficiently small to resolve the proteins
Discontinuous:
 Different buffer ions are present in the gel and electrode reservoirs
Sample is loaded on to large pore-size gel called the stacking gel
polymerized on top of the resolving gel
25
 Advantage: Large volumes of dilute samples can
be applied and gives good resolution as proteins
are concentrated into narrow zones (or stacks)
during migration through the stacking gel prior to
their separation in the resolving gel.
26
Electrophoresis Buffers
Tris HCl pH 6.8 = Stacking gel
Tris HCl pH 8.8 = Resolving gel
Tris-glycine pH 8.3 = Running buffer
Tris-HCl pH 6.8 & glycerol = Sample diluting buffer
27
28
Formation of an ion front:
Cl = Leading ion
Gly = Trailing ion
29
What happens to the proteins?
Proteins have mobilities between those of Gly and Cl-.
30
In separating gel
Glycine mobility increases, becomes greater than protein
mobility, but still slower than Cl-.
• Protein sample, now in a narrow band, encounters both the increase in pH and
decrease in pore size.
• Increase in pH would tend to increase electrophoretic mobility, but smaller pores
decrease mobility.
• Relative rate of movement of ions in lower gel is chloride > gly > protein.
• Proteins separate based on charge/mass ratio and on size and shape parameters.
31
Slab gel Electrophoresis
32
Gel preparation and sample loading
33
34
Visualizing & Analyzing the Gel
35
Staining the Gels
Normally the target molecules of the electrophoretic process
must be visualized in a staining process after the separation.
(1) Direct Staining → Coomassie brilliant blue R250
(2) Silver Staining → Silver Nitrate
36
Direct Staining → Coomassie brilliant blue
• Normally Coomassie dye is used for staining proteins after SDS and native
electrophoresis.
• A commonly used stain for detecting proteins in polyacrylamide gels is 0.1%
Coomassie Blue dye in 50% methanol, 10% glacial acetic acid. Acidified
methanol precipitates the proteins
• The dye actually penetrates the entire gel, however it only binds permanently
to the proteins.
• Excess dye is washed out by 'destaining' with acetic acid/methanol, also with
agitation. Properly stained/destained gels should display a pattern of blue
protein bands against a clear background. The gels can be dried down or
photographed for later analysis and documentation.
37
Silver Staining → Silver Nitrate
• Silver staining is generally used when detection of very faint proteins
is necessary.
• Very sensitive. Silver staining methods are about 10-100 times more
sensitive than various Coomassie Blue staining techniques.
• The most commonly used source of Ag+ is AgNO3. However, the use
of silver nitrate usually gives high background but the use of various
types of sensitizers such as thiosulfate ions has been shown to decrease
the background and increase detection sensitivity.
• After fixing the proteins by methanol/acetic acid the sensitizer is added
to the gel which chelates the silver. Silver ions binds to the proteins
and can easily and specifically be reduced to solid silver (Ag) by a
reducing agent such as formaldehyde under alkaline conditions (e.g.
Na2CO3). This reaction will stain the protein bands giving them a
brown color. The reaction is stopped by adding acid.
38
Step
Reagent
Volume
Time
1 Fixing
40%(v/v) Ethanol / 10% Acetic Acid (v/v)
(80 ml EtOH/20 ml HAc)
(place gel onto the solution in a glas tray, gel swims
surface side down)
200 ml
30 min
2
Incubation
30% Ethanol; 0.2% Sodium Thiosulphate;0.5 mol/l
sodium acetate; 0.125 % Glutardialdehyd (w/v)
(60 ml EtOH/0.4 g Thiosulfate/13.6 g NaAc/1 ml
Glutardial.)
200 ml
30 min
3-5
Washing
H2O dist (place gel on the glas tray bottom surface
side up)
3 x 200
ml
3x5
min
6 Silvering
0.25% AgNO3 /0.015% Formaldehyde (w/v)
(0.5 g AgNO3/80 µl Formaldehyde)
200 ml
20 min
7,8
Washing
H2O dist
2 x 200
ml
2x1
min
10
Developing
2.5 % Na2CO3 / 0.015% Formaldehyde
(5 g NaCO3/80 µl Formaldehyde)
2 x 200
ml
3-5
min
11
Stopping
Preserving
10% HAc, 10% Glycerol
(20 ml HAc/20 ml Glycerol)
200 ml
20 min
Drying
air dry , on the support-film
then roll on a polyester-sheet
---
16 h
39
40
Analysis of bands
41
Applications
• Estimated molecular masses and relative abundance of
unknown polypeptides in a complex mixture
• Patterns of bands that suggest presence of isoenzymes or
specific complex proteins
• Effectiveness of a separation procedure during cell/tissue
fractionation
• Effectiveness of a procedure to purify specific organelles,
proteins, or polypeptides
• Condition of a preparation such as extent to which proteins
have degraded
• Similarity of one preparation to another
• Changes in gene expression during developmental stages
or resulting from experimental intervention
42
Troubleshooting
43
Critical Parameters and Troubleshooting
• It is important to use only high-quality electrophoresis-grade reagents when
running the gels. Acrylamide and bisacrylamide both break down in solution
to acrylic acid, which affects the mobility of molecules through the gel matrix.
• Acrylamide solutions should be protected from light and should not be stored
for more than a few months.
• Acrylamide monomer is a potent neurotoxin. Do not mouth pipette acrylamide
solutions, and wear gloves when handling unpolymerised solutions
• Ammonium persulfate should be made fresh.
• Clean plates are also essential in order to avoid the introduction of bubbles
into the gel when pouring.
44
Smiling Bands
The lanes in the center of an overheated gel run faster than the lanes at the
sides. This is caused by uneven dissipation of heat by the gel.
There are several ways to avoid smiling, the simplest of which is to run the
gel at lower voltage. An alternative is to use an apparatus that incorporates a
mechanism such as a metal plate to disperse heat evenly throughout the gel,
or an active cooling mechanism
45
High Sample concentration
46
Too much Salt / Buffer in the Sample
After the samples were filled in the sample-wells and the
voltage is applied sample jump out of its slot and run over
the stacking gel. This happens when there are too much
ions in the sample volumes. It can also happen that ´salty
samples´ leave their slots even without an applied electric
field.
To overcome this, urea is added. If urea is not the solution
then the sample is passed through a special desalting
column
47
Smeared Bands
Smearing can have a variety of causes, but most
commonly it is due to an unevenly poured
acrylamide mixture or due to gross overloading
of protein.
48
Western Blotting
• Introduction
• Basic Concepts and Method
• Troubleshooting
49
Introduction
50
• The term “blotting” refers to the transfer of biological
samples from a gel to a membrane and their subsequent
detection on the surface of the membrane.
• Western blotting (also called immunoblotting because an
antibody is used to specifically detect its antigen) is now a
routine technique for protein analysis.
• The specificity of the antibody-antigen interaction enables
a single protein to be identified in the midst of a complex
protein mixture.
• Western blotting is commonly used to positively identify a
specific protein in a complex mixture and to obtain
qualitative and semiquantitative data about that protein.
51
Western blotting evolved from Southern blotting, invented by
Edwin Southern at University of Edinburgh in 1975, then
Northern blotting, invented by George Stark's Stanford group
in 1977.
1079: Neal Burnette’s group (at Fred Hutchinson), Harry
Towbin's group (in Switzerland) and George Stark's group (at
Stanford)
Stark's group published first. Towbin's group developed what
appears to be the most common method, including the
electrophoretic transfer method and buffers, as well as the use
of secondary antibodies. Burnette gave the technique the
name among other modifications.
52
Basic Concepts and Method
53
•
Proteins are separated by gel electrophoresis, usually
SDS-PAGE.
•
The proteins are transferred to a sheet of special blotting
paper called nitrocellulose.
Transfer may be done by any of the following methods:
1. Wet Transfer
2. Semi Dry Transfer
3. Dry Transfer
54
Wet Transfer
55
Semi Dry Transfer
56
Dry Transfer
57
58
• The blot is incubated with a generic protein (such as milk
proteins) to bind to any remaining sticky places on the
nitrocellulose.
• An antibody is then added to the solution which is able to bind
to its specific protein.
59
Detection
60
• The antibody has an enzyme (e.g. alkaline phosphatase or
horseradish peroxidase or a fluorescent probe) or dye
attached to it which cannot be seen at this time.
• The location of the antibody is revealed by incubating it
with a colorless substrate that the attached enzyme converts
to a colored product that can be seen and photographed.
61
62
63
Troubleshooting
64
High Background
Antibody concentration too high
Insufficient blocking
Cross reactivity of antibody with the proteins in the blocking buffer
Membrane got dry
Contamination of buffer
Contaminated equipments
65
Weak or no signal
Proteins did not transfer properly
Insufficient binding to the membrane
Insufficient amount of antibody
Insufficient amount of antigen
Antigen masked by blocking buffer
Presence of azide (in case of HRP detection)
Reaction time is too short
Substrate / Enzyme has lost activity
66
Non specific binding
Antibody concentration too high
Polyclonal antibody used as secondary antibody
Diffused bands
Antibody concentration too high
Too much sample loaded
67
• http://www.currentprotocols.com/WileyCD
A/CurPro3Video/videoId-694565070.html
68
Further Reading
Physical Biochemistry : Applications to Biochemistry and Molecular Biology
by David Friefielder
2nd Edition
Publisher: New York : W.H. Freeman, ©1982
Gel Electrophoresis of Proteins: A Practical Approach
by BD Hames & D Rickwood
Publisher: IRL Press Ltd. Oxford, Washington DC ©1981
Introduction to Biotechnology by W.J. Thieman and M.A. Palladino.
Pearson & Benjamin Cummings
69