BCHEM 264

Lecture 5
July 18, 2022

1
 Gel Electrophoresis
• Gel electrophoresis is a broad subject encompassing many different
techniques
• There are many variations of gel electrophoresis of proteins
• Gel electrophoresis reveals information about molecular weight,
subunit structure, purity, and charges on proteins
• The most common use of gel electrophoresis is preparative and
qualitative analysis of complex mixtures of proteins
• When combined with microanalytical methods and sensitive, linear
image analysis systems, gel electrophoresis can be used for
quantitative studies of proteins
• Gel electrophoresis provides the highest resolution of all methods
available for separating proteins. Polypeptides differing in
molecular weight by as little as a few hundreds of Daltons, and
proteins which differ by less than 0.1 pH unit in their isoelectric
points are routinely resolved on electrophoresis gels.

2
 Gel Electrophoresis
• Gel electrophoresis of proteins produce pure
proteins for further work such as:
• Determination of the protein’s amino acid sequence
• Use of the protein in fingerprinting studies to reveal
polymorphisms and evolutionary relationships, such
as in genetic diversity studies
• A pure protein is required to elucidate a protein’s
tertiary structure, hence its biochemical function by
means of X-ray crystallography

3
 Websites for electrophoresis equipment and
knowhow
• www.cleaverscientific.com
• www.biorad.com
• www.cbdscientific.com

4
 Variations in gel electrophoresis of proteins

• Tube vs. slab gel electrophoresis

• One dimensional (1D) vs. two dimensional (2D) gel
electrophoresis
• Native vs. denaturing electrophoresis
• Continuous vs. discontinuous gel systems

5
 Tube vs. slab gel electrophoresis
• Acrylamide can be polymerized into any desired
shape
• Two shapes in common use in electrophoresis
– Tube Gels : polymerize in glass tubing to give
cylindrical shaped gel
– Slab Gels: polymerize between glass plates to
give a rectangular gel

6
Tube gel
electrophoresis
•Polymerization is carried out in glass
tubes of dimensions 7 mm × 10 cm in
length
•For isoelectric focusing (I.E.F.) gels
and 1st dimension of 2-D
electrophoresis
•Advantages of tube gels:
•Tube gels are easy to load
•Technique requires minimum use of
apparatus
•Disadvantages:
•Only one sample can be run per tube,
Apparatus includes solid
however, there are many places for 12,
vacutainer stoppers for
24, etc. tubes per vacutainer
variable size tubes. Has a
•Inaccurate comparison among cooling jacket for temperature
different samples as different tubes control and interlocking safety
would have different conditions cover 7
 Slab Gel Electrophoresis
• Vertical gel slab sizes range from small (2 cm × 3 cm)
to large (15 cm × 18 cm)
• Advantages of small vertical gels
– Require less time and reagents than large gels
– They are good for rapid screening of samples
• Advantages of large vertical gels
– Provide better resolution
– Good for separating proteins of similar sizes , as well as a
large number of proteins
• Precast slab gels are commercially available for
techniques such as SDS-PAGE, native gels, and iso-
electric focusing

8
Precast protein gels for
SDS-PAGE
A Thermo Scientific Pierce
Precise Protein Gel
Cassette. The plastic
cassette contains a mini-
gel that is 1 mm thick.
Dividers along the top
provide 10 wells for
loading protein samples
and molecular weight
markers (ladder).
Ordinarily, protein bands
would not be visible until
after electrophoresis they
are disassembled from the
cassette and and stained.

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 Slab gel electrophoresis

• Slab gels offer the following advantages

– Permit analysis of up to 20 samples on a single gel
slab having identical gel conditions
• Applications of vertical gel slabs
– For routine analysis of proteins
– For separation of DNA fragments during DNA
sequence analysis

10
 One dimensional vs. two dimensional
electrophoresis
• Electrophoresis can be one dimensional (1D) (i.e. one plane
of separation) or two dimensional (2D)
• One dimensional electrophoresis is used for most routine
protein and nucleic acid separations
• It is also used for comparative analysis of multiple samples
• Gel sizes range from 2 cm × 3 cm (tiny) to 15 cm × 18 cm
(large format). The most popular size (8 cm × 10 cm) is
usually referred to as a "Mini-gel“
• An example of a one-dimensional separation of proteins is
shown below. In this configuration, the protein pattern is one
of multiple bands with each band containing one protein or a
limited number of proteins with similar molecular weights

11
 1 2 3 4 5 6 7
A typical analytical SDS-PAGE
gel showing muscle proteins
from five fish varieties and a
control from rabbit muscle
extract. Samples were
separated by SDS-PAGE in a
precast mid-size-gel and
stained with colloidal CBB G-
250.
Lanes are from left to right:
marker proteins, shark,
salmon, trout, catfish,
sturgeon, and rabbit (actin
and myosin). The salmon and
trout patterns (lanes 4 and 5)
are very similar, as expected
given the close evolutionary
relationship between the two
species. All of the fish samples
appear to contain muscle
proteins similar to those of
rabbit. 12
 One Dimension vs. Two dimension
Electrophoresis
• Samples that are loaded in adjacent wells and
electrophoresed together are easily compared to each other
after staining or some other detection system
• The intensity of staining and "thickness" of protein bands are
indicative of their relative abundance
• The position (distance) of bands measured from the well in
each lane indicates the relative size in Daltons for protein or
in base pairs for nucleic acids. Distance moved or mobility is
a function of size, shape, and charge.
• Below, we find an electrophoregram of protein lanes and
bands in 1D SDS-PAGE. Photograph of a mini-gel after
removal from the cassette and staining with Coomassie dye
(Thermo Scientific GelCode Blue Stain Reagent). The mini-gel
has ten lanes, each containing many protein bands of varying
abundance

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Precast protein gels for
SDS-PAGE

A Thermo Scientific Pierce

Precise Protein Gel
Cassette. The plastic
cassette contains a mini-
gel that is 1 mm thick.
Dividers along the top
provide 10 wells for
loading protein samples
and molecular weight
markers (ladder). A
common ladder for SDS-
PAGE is PageRuler
Prestained Ladder. This
was loaded in lanes 5 and
8. Another Ladder was
loaded in lanes 1 and 6

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 One dimensional vs. two dimensional
electrophoresis

• Two dimensional separation of proteins is

used for fingerprinting, and when properly
constructed can be extremely accurate in
resolving all of the proteins present within a
cell (greater than 1,500).
• Two-dimensional separation of nucleic acids is
used to resolve over 3,000 transcripts in gene
expression studies

15
 Native vs. denaturing gel electrophoresis
• Native: electrophoresis is carried out in the absence of denaturing
agents such as heat, reducing agents
• For proteins: the reducing agents, β-mercaptoethanol and
Dithiothreitol remove the disulfide linkages between polypeptides.
Sodium dodecyl sulfate plus heat denature the tertiary structure
and unwind the protein into a single fairly straight polypeptide
• For nucleic acids, urea and formamide denature by forming
hydrogen bonds with the bases and occupying the H-bond sites. H-
bonds can therefore not form between the bases of the two
strands. Again, urea and formamide lower the melting temperature
of double stranded DNA and allow denaturation to proceed at low
temperature.

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 Native vs. denaturing gel electrophoresis

• The buffer system in native gel electrophoresis is nonreducing

and nondenaturing and maintains the protein’s secondary,
tertiary, and quaternary structures, as well as its native charge
density; and the native structure of dsDNA
• Separation of proteins and nucleic acids in nondenaturing gels
is therefore based on their charge, size, and shape,
particularly, their charge-to-mass ratio
Separation of proteins by native gels electrophoresis
• Migration of proteins in native gels is governed by the intrinsic
charge (the combination of amino acids and post-translational
modifications, e.g., addition of phosphates, sialic acid, etc.) on
the protein at the pH of the running buffer.
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 Native vs. denaturing gel electrophoresis
• Migration of the protein also depends on the hydrodynamic
size of the unfolded protein, hence, the conformation of the
protein.
• A higher mobility is expected for more compact
conformations, and lower mobility for larger structures like
oligomers
• Native –PAGE for most proteins may be carried out at
different pH for acidic proteins, basic proteins, and near-
neutral proteins
• The idea is to prevent acid or alkaline denaturation of the
protein so that the conformation, self aggregation, and
binding of other proteins or compounds is preserved and may
be studied
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 Native Gel Electrophoresis
• There are two methods:
(1) Nondenaturing polyacrylamide gel electrophoresis of proteins
(2) Nondenaturing polyacrylamide gel electrophoresis of nucleic acids
• For proteins, the nondenaturing gel retains the activity of native
proteins, because the folded structure, charge, shape and size are
preserved allowing the structure of the protein to drive its mobility
in the gel. For DNA, the small pore sizes of the polyacrylamide gel is
used for small amplified DNA sequences in fingerprinting (10 bp to
3,000 bp)
• Native PAGE does not provide direct measurement of molecular
weight, but can be used to estimate protein charge or subunit
composition

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 Native Gel Electrophoresis
• Electrophoretic migration is ensured by running the
electrophoresis in alkaline running buffers (usually, pH 8-9 of
the Laemmli system). At this pH most proteins carry a net
negative charge and the magnitude of the charge is
influenced by the intrinsic charges on the protein.
• The higher the negative charge density (more charges per
molecular mass), the faster the protein migrates.
• During migration, the proteins encounter the frictional
resistance of the gel matrix which creates a sieving effect
• The proteins therefore migrate according to their size and
three-dimensional shape. Small proteins face only a small
frictional force, while larger proteins face a larger frictional
force. Thus native PAGE separates proteins based on both
charge and mass. 20
 Native Gel Electrophoresis
• Because no denaturants are used in native PAGE,
subunit interactions within a multimeric protein are
generally retained and information can be gained
about the quaternary structure.
• In addition, some proteins retain their enzymatic
activity (function) following separation by native
PAGE. Thus, this technique may be used for
preparation of purified, active proteins.

21
 Native Gel Electrophoresis

• Native PAGE is good for separating proteins of

identical molecular weight but different charges
which cannot be resolved with SDS-PAGE.
• Because proteins on native PAGE usually retain their
activity, the technique can be used to detect
enzymes in their biologically active form

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 Native Gel Electrophoresis of proteins
• Separation of proteins on native gels depends on the mobility
of the protein
• Mobility on native gels is a function of charge, mass, and
isoelectric point of the protein and is governed by the
equation
log R m  log Y0  K R T.....(1)
where Rm = relative mobility, normalized to the tracking dye
front; Rm is analogous to retention factor
Y0 = relative mobility of the protein in free solution, i.e., in the
absence of any sieving matrix or a gel. Yo is related to the
charge on a protein
KR = retardation coefficient or mechanical resistance of the
gel. It measures the extent to which a gel matrix impedes
mobility. It is related to size or MW of the protein
T = % monomer (acrylamide) of the gel matrix

23
 Native Gel Electrophoresis

• In the presence of SDS, an anionic detergent, all

proteins have the same charge-to-mass ratio and
identical relative mobilities in free solution, Y0
• In such a case, a simple relationship exists between
Rm and KR when %T is known. In a free solution
therefore, SDS-treated proteins will migrate at the
same speed. Knowing KR means that one can
calculate mobility
• KR is directly related to molecular weight and so
molecular weight can be calculated from KR
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 Native Gel Electrophoresis

• In native gels, the situation is more complicated.

Both Y0 and KR can vary between proteins. Y0 is
related to the charge, while KR varies with molecular
mass or size of the protein.
• Y0 and KR are obtained from graphs known as
Ferguson Plots
• To plot such graphs, protein mixtures and standards
are separated on native polyacrylamide gels of
varying percentages of acrylamide (%T)

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 Native Gel Electrophoresis

• The distance travelled by a protein, log Rm , is plotted

against %T.
log R m  log Y0  K R T.....(1)
• From equation (1), the graph would have a slope of
-KR and a Y intercept (when %T = 0) of Yo

• Comparison with standards of known charge and size

allows determination of the charge and molecular
weight of the unknown samples.

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 Native Gel Electrophoresis
Ferguson Plots

Three proteins of the same charge Three proteins of the same mass,
(Y0) but different masses i.e., slope (KR) but different charges

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 Native Gradient PAGE

• Gradient gels offer a simpler method of

interpretation of native gels.
• Gradient gel is made with increasing
acrylamide concentration along the length of
the gel, hence pore size gradually decreases
from top of gel to the bottom
• As proteins migrate they enter smaller pore
size range and their mobility decreases.
• Eventually, each protein reaches its "pore-
limit", at which point it stops migrating.
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 Native Gradient PAGE

• At the pore limit, there is no movement and the

relative positions are a direct reflection of their
molecular weight.
• A plot of log MW against the distance moved by each
protein (Rm ) gives a sigmoid curve. Within the linear
gradient range of this curve, log MW is proportional
to log Rm over a wide range of molecular weights

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 Native Gradient PAGE
• Proteins separated by native gradient gels may be visualized
by the general protein staining reagents as well as activity
stains where the protein is likely to be an enzyme. These are
stains which enable visualization of enzyme activity.
• Activity staining involves the use of chromogenic or
chemiluminescent compound which reacts with the enzyme
(the protein separated) to deposit colored, insoluble
compound in the gel, or a luminous product, respectively.
• If the reaction is a direct involvement with the enzyme to
produce color, we have a positive stain. If the colored
compound is formed in a reaction inhibited by the enzyme,
then we have a negative staining technique

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 Native gel electrophoresis
for DNA
Nondenaturing polyacrylamide gel
electrophoresis is used for DNA
fingerprinting in marker systems such as
RAPDs and SSRs to examine small
fragment sizes (10-300 bp)
amplified by the primers
RAPDs = Random Amplified
Polymorphic DNA sequences
SSRs = Simple Sequence Repeats
It is recommended that the researcher
begins with 12% of 29:1 acrylamide/bis-
acrylamide as the starting point
Concentration may be reduced (e.g., to
8%) or increased (e.g., to 12%) for
larger or smaller fragments,
respectively. For nondenaturing gels,
Tris-glycine buffer (25 mM Trizma-base,
192 mM glycine) may be used.
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 RAPDs on agarose gel
Nondenaturing gels for
DNA
Run the gels at constant
volatge of 250 V for 2-5 h,
depending on the
acrylamide concentration.
Generally it takes 2 h for
8%, and 3 h for 12%, 5 h
for 16% gels when the
tracking dyes,
bromophenol blue and
xylene cyanol reach the SSRs on
bottom of the gel PAGE

32
 Buffers for electrophoresis

• The electric current in an electrophoresis cell is

carried largely by ions in the buffer
• Note that proteins constitute only a small portion of
this current
• The buffer system also:
-maintains the desired pH for native systems and
for SDS-PAGE
-provides a medium for heat dissipation

33
 • Schematic for continuous gel
Buffers for electrophoresis
electrophoresis
Two classes of buffer
systems are commonly
used in electrophoresis:
continuous or
discontinuous
Continuous buffer systems
Continuous systems use
one and the same buffer
PAGE
ions at constant pH in the
gel, sample, and electrode
reservoirs/tank. A single
separating/resolving gel is
used Agarose gel

34
 Continuous gel buffers

• In continuous systems, the sample is loaded directly on the

gel in which separation will take place, like a resolving gel.
• Continuous gels are easy to prepare and give adequate
resolution for some applications, however, bands tend to be
broader and resolution consequently poorer
• To improve on resolution, the strength of the sample buffer is
diluted. Dilution leads to decreased buffer ions, a decrease in
conductivity and hence localized voltage drop across the
sample
• Voltage drop in the sample helps drive proteins into the gel

35
 Continuous gel buffers
• As proteins migrate through the pores of the gel they are
separated on the basis of electrophoretic mobility differences.
• The width of bands formed is highly dependent on the height
of the applied sample volume
• To prevent smearing of bands, small sample volumes (5-15
microliter) must be applied. This can be achieved by using
highly concentrated samples
• In continuous systems, molecular charge density and gel pore
size are the only factors that control mobility and resolution
• Larger molecules are better resolved on continuous buffer
systems than small molecules because of a small difference
between their free solution mobility and their mobility in a
gel. Continuous buffer systems work best with highly
concentrated samples
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 Buffers for electrophoresis
• Almost any buffer can be used for continuous gel
electrophoresis, but for best results, solutions of relatively low
ionic strengths are used
• Buffers of low ionic strength keep heat generation at a
minimum. On the other hand, protein aggregation may occur
if the ionic strength is too low.
• Buffers for DNA/RNA include TAE (Tris-Acetate-EDTA) or
TBE(Tris-Borate-EDTA) . TAE offers a lower buffering capacity,
hence conducts lower current and requires more time, but
offers better separation.
• Different buffers exist for different proteins : TRIS-glycine,
TRIS-tricine, etc.
• Concentrations of electrophoresis buffers are in the range of
from 0.01 to 0.1 M
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 Buffers for electrophoresis
Discontinuous buffer systems
• Discontinuous buffer systems employ different buffers and
different pH for tank and gel, and often two different buffers
within the two gel systems
• Discontinuous systems are designed to enhance the sharpness
of the separated bands by sharpening the starting zones. This is
achieved in a process called "stacking“. It involves
concentrating protein samples into a very narrow and tight
zone prior to separation. It results in improved band sharpness
and resolution.
• Stacking is an electrochemical phenomenon that establishes an
ion gradient in the early stage of electrophoresis on the basis
of mobility differences between (1) proteins, (2) leading buffer
ions and (3)trailing buffer ions.
• Differences in mobilities cause all the proteins to focus into a
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single sharp band
 Discontinuous Gel Electrophoresis

A discontinuous gel system

Electrode/tank
buffer

Stacking gel buffer,

lower
concentration gel
with large pores

Resolving gel
buffer, higher
concentration gel
with smaller pores

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 History of discontinuous gel electrophoresis
• Starch gels were first used in protein separation
• In 1964, starch gels were replaced by polyacrylamide
gels (Ornstein, 1964; Davis, 1964)
• Shapiro et al. (1967) developed SDS-PAGE by
including sodium dodecyl sulfate (SDS) to the
polyacrylamide gel as well as the sample
• Laemmli (1970) developed protocols ( the
discontinuous system) to greatly improve the
resolution and power of protein identification by
SDS-PAGE

40
 History of discontinuous gel electrophoresis
• Laemmli did two things:
• (a) He introduced the discontinuous gel system having different
pore sizes:
• (i) Stacking gel: low acrylamide concentration (usually 3%), so no
sieving effect; low pH 6.8
• (ii) Resolving (separating) gel: high acrylamide concentration
(usually 10%), sieving effect, proteins move freely into the resolving
gel; high pH 8.8
• (b) He added glycine and TRIS to improve the ionic strengths and pH
of the buffers
• Three buffer systems were produced (1) Stacking gel, pH 6.8 TRIS-
HCl buffer; (2) Resolving gel pH 8.8 TRIS-HCl buffer; (3) Tank or
electrode buffer pH 8.3 TRIS-glycine
• Control of the charge state of glycine by the different buffers drives
the stacking process
41
 Discontinuous gel electrophoresis

• Glycine can exist in three charge states, namely,

positive, neutral or negative, depending on pH of the
medium shown below

42
 Discontinuous gel electrophoresis

• At very acidic pH (below pKa1) the amino acid will

have an overall +ve charge and at very basic pH
(above pKa2 ) the amino acid will have an overall -ve
charge
• The isoelectric point is average of the two pKas,
i.e. pI = 1/2 (pKa1 + pKa2)= ½(2.34 + 9.6), pI = 5.97

43
 Discontinuous gel electrophoresis

• The stacking gel works like this: because pI of glycine

is 5.97, in the tank buffer with pH 8.3, glycine will be
negatively charged
• When power is turned on, the negatively-charged
glycine ions in the tank buffer are forced to enter the
stacking gel, where the pH is 6.8.
• At this relatively lower pH close to the pI, glycine
switches predominantly to the zwitterion state
causing them to move very slowly in the electric field

44
 Discontinuous gel electrophoresis
• In contrast, the Cl- ions (from Tris-HCl) in the stacking gel
with their small size and negative charge move much
more quickly in the electric field and form an ion front
(leading ion) that migrates ahead of the almost neutral to
positive glycine (trailing ion)
• The separation of Cl- from the TRIS counter-ion (which is
now moving toward the anode) creates a highly mobile
Cl- front and a slowly migrating glycine with a narrow
zone of steep voltage gradient between them
• The gradient pulls along the glycine but remains behind
the Cl-

45
 Discontinuous gel electrophoresis

• The abrupt voltage drop causes the proteins in the

gel sample having an electrophoretic mobility that is
intermediate between the extreme of the mobility of
the glycine and Cl-, to move rapidly (sweeping) to fill
the gap and prevent a break in the circuit (Kolrausch
discontinuity)
• By this, proteins are concentrated (stacked) into a
tight band between the Cl- and glycine fronts,
thereby improving resolution

46
 Discontinuous buffer systems

• The width of the stack therefore becomes very thin

and compact, having about 100 µm thickness and
with protein concentrations of about 100 mg/ml
• Electrophoretic stacking concentrates proteins into
regions narrower than can be achieved by
mechanical means. This has the effect of minimizing
spreading of overall band widths and increasing
resolution.
• In order to allow the stack to develop, gels used with
discontinuous systems are usually divided into two
distinct segments.

47
 Discontinuous buffer systems

• The smaller, upper portion is called the stacking gel.

It is cast with appreciably larger pores than the lower
resolving gel (or separating gel) and serves mainly as
an anticonvective medium during the stacking
process. In the stacking gel, no sieving effect occurs.
• Separation takes place in the resolving gel, which has
pores of roughly the same size as the proteins of
interest. Once proteins enter the resolving gel their
migration rates are slowed by the sieving effect of
the small pores
48
 Discontinuous buffer systems

• In the resolving gel, the trailing ions pass the proteins and
electrophoresis continues in the environment supplied by
the electrode buffer. The proteins are said to become
“unstacked” in the resolving gel. They separate there on
the basis of size and charge
• The electrophoresis monitored and timed by means of the
dye front. Migration of the buffer front as it moves through
the gel can be followed by the change in refractive index
between the regions containing the leading and trailing
ions. Addition of a tracking dye that moves with the buffer
front aids in visualization of the protein front
• The major advantage of a discontinuous buffer system is
increased resolution and sharpness of the sample band
49
 Gel electrophoresis of proteins
Many techniques are available
• Sodium-dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
 Native (buffer) gels
 Gradient gels
• Iso-electric focusing
• Two-dimensional polyacrylamide gel electrophoresis
• Cellulose acetate electrophoresis
• Western blotting

50
 SDS-PAGE
• SDS-PAGE is the most widely used method for
analyzing protein mixtures qualitatively
• It is an example of denaturing gel electrophoresis
• A demerit of native PAGE is that the native structure
of a protein affects mobility in a gel matrix such that
the rate of migration is not a true estimate of the
charge:mass ratio nor an accurate reflection of the
molecular weight
• In their native structure, proteins fold into a variety
of shapes to produce compact or elongated forms.
Nature of folding depends on the amino acid
sequence of the protein
• To overcome structural effects on mobility,
denaturation of the protein is carried out
51
 SDS-PAGE
• Denaturation allows separation of proteins on a true
charge/mass ratio basis. It also separates proteins
into their individual subunits and permits analysis of
large, complex protein aggregates
• Sodium dodecyl sulfate (also called sodium lauryl
sulfate) is the most common denaturant used. Other
denaturants are Triton X-100 and β-octylglucoside.
Both are nonionic.
• The molecular formula of SDS is given below:

52
 SDS-PAGE

• MW: 288.38 (CH3  (CH 2 )10  CH 2OSO3 Na  )

• SDS denatures proteins by binding to the protein chain with
its hydrocarbon tail, exposing normally buried regions and
coating the protein chain with negatively charged surfactant
molecules. SDS disrupts most noncovalent bonds, thereby
decreasing protein folding.
• Proteins bind the SDS detergent uniformly along their length
to a level of 1.4 g SDS/g protein or one SDS to 2 amino acid
residues. This creates a charge/mass ratio which is uniform or
consistent between proteins.
• Each protein in the mixture is therefore fully denatured by this
treatment and opens up into a rod-shaped structure with a
series of negatively charged SDS molecules along the
polypeptide chain.

53
 SDS-PAGE

• For this reason, separation on a polyacrylamide gel in

the presence of SDS occurs by mass alone, hence the
sieving effect of the gel governs the separation.

• SDS PAGE offers a rapid and relatively accurate way

to determine protein molecular weights. Molecular
mass determined by SDS-PAGE is usually accurate
within 5 - 10% error rate

54
 SDS-PAGE
• An exception: some proteins, such as, histones may not be
fully denatured and may retain enough secondary structure or
contain sufficient charged groups to migrate anomalously so
that molecular weight will not be a direct reflection of their
mobility. In such a case, Triton X-100 combined with urea and
acetic acid are used to enhance denaturation
Denaturation of proteins for SDS-PAGE
• Complete dissolution of sample is required. Undissolved
particles present in the sample will lead to clogging of the gel
and smearing of the protein band from the well to the end of
the gel.
• Complete denaturation of protein sample is required.
Incomplete denaturation will not fully saturate the proteins
with SDS and will lead to blurred bands or altered mobilities
55
Structure and function of
detergent micelles. The
three commonly used
detergents are sodium
dodecyl sulfate (SDS), an
anionic detergent; Triton X-
100 and β- octylglucoside
are two nonionic
detergents. Triton X-100 is
a mixture of compounds in
which the region in
brackets is repeated 9 or
10 times. The hydrophobic
portion of each detergent
is shown in yellow, and the
hydrophilic portion is
shown in orange.

56
 SDS-PAGE
• SDS is a strong detergent which completely dissolves many cells and
tissues by heating to 95°C in loading buffer
• To solubilize more difficult samples such as plant tissue, a stronger
loading buffer, containing more SDS and dithiothreitol (DTT) at high
pH is used
• Reagents: sample buffer for tissue culture cells and soft tissue
0.5M Tris-HCl, pH 6.8
4.4% SDS
300 mM Mercaptoethanol
10mg/ml Bromophenol blue
• Mercaptoethanol reduces the disulfide bridges holding together the
protein tertiary and quaternary structure
• Mix sample with an equal volume of 2× sample buffer and heat to
95° C for 10 min, cool to room temperature and centrifuge at
14,000 rpm for 5 min in a microcentrifuge to remove all particles.
Load the gel 57
 SDS-PAGE
• Reagents: sample buffer for plant and hard tissue – CHES buffer
1% CHES (N-cyclohexyl-2-aminoethanesulfonic acid), adjust pH
to 9.5 with NaOH
2% SDS
1% DTT
10% glycerol
Can be stored at -20° C up to 6 months
Homogenize sample in 5-15 volumes of CHES buffer in a homogenizer
and heat to 95° C for 10 min, cool to room temperature and
centrifuge at 14,000 rpm for 15 minutes. Load the gel
The lipopolysaccharide (LPS) layer of the capsule of yeasts, fungi, and
Gram negative bacteria cell walls requires enzymatic digestion with
lysozyme or zymolyase prior to homogenization

58
 SDS-PAGE
• In SDS gel electrophoresis of samples SDS imparts identical charge:mass
ratio to all proteins
• The rod-like structure remains stable as any attempt that tends to fold up
the protein chain would result in repulsion between negative charges on
different parts of the protein chain, returning the conformation back to
the rod shape

SDS attaches to protein by its

hydrophobic chains.
Negatively charged SO42-
groups are exposed to the
medium

59
 SDS-PAGE

• The sample buffer also contains the following:

• An ionizable tracking dye, usually bromophenol blue
These allow the electrophoretic run to be monitored
so samples do not run beyond the plate
• Sucrose or glycerol, which gives the sample solution
some density, allows the sample to settle easily
through the electrophoresis buffer to the bottom
when injected into the well

60
Preparation of • SDS-PAGE system was developed by
Denaturing Protein Laemmli (article in Nature, 1970).
Gels • The classic Laemmli SDS-PAGE
method uses a discontinuous gel
system
• Discontinuous buffer systems use
different buffers for tank and gel,
and often two different buffers
within the gel, with a third buffer in
U. K. Laemmli the tank.
Professeur Emeritus
University of Geneva, Switzerland. • Because discontinuous systems
Department of Molecular Biology concentrate, or stack the protein
samples into a very narrow zone
prior to separation, two gels are
prepared: stacking gel and resolving
gel
61
 Denaturing gels
• The upper stacking gel is prepared with low
acrylamide percentage (i.e. large pore size to allow
proteins to move freely and concentrate into a tight
band) and low pH of 6.8 [low %T, low pH]
• The lower resolving gel is prepared with high
acrylamide percentage (means smaller pores) with a
pH of 8.8 [high %T, high pH]
• Both gels contain only Cl- as the mobile anion. The
tank buffer has glycine as its anion, at a pH of 8.3.

62
SDS-PAGE

Photograph showing samples being

loaded into the wells of an SDS-
PAGE minigel. Six wells that have
been loaded can be identified by the
blue dye (bromophenol blue) added
to the loading buffer

63
 Gel electrophoresis apparatus and reagents
• Many variations in apparatus available from different manufacturers:
Bio-Rad PROTEAN, Mini Plate, Thermoscientific, Cleaver Scientific, etc.
Apparatus
1) A set of 2 glass plates: a rectangular plate, a short notched plate
2) Spacer (1mm thick): 2 are needed, one for each opposite side. You can
use a flexible tape for this
3) Gel casting system (casting frame to sandwich glass plate; casting stand
for glass plates, or casting frame)
4) Clamping frame and electrode assembly for precast gel
5) Comb (1 mm thick) for glass plates; precast gels already have comb
attached (gel cassette)
6) Electrophoresis tank or Gel box with lid; lid brings the power from the
power supply
7) Micropipettes with slender tips

64
 Two spacers
Rectangular and notched plate Glass plates with two
spacers attached

Casting frame (bottom) and Combs

casting stand (up)
65
 Casting frame with comb Gel cassette
fitted to a casting stand

Buffer dam
Clamping frame and electrode assembly
with gel cassette in place (comb removed
to show numbered wells
66
Clamping frame and electrode assembly with
casting gel or gel cassette in front and buffer
Dam behind; has two ears with red and black color
coding

Gel box or electrophoresis

tank with lid 67
Pipette tips with slender Normal pipette tips
ends for loading a gel

Power pack

Gel box connected to power pack 68

 Reagents for vertical gel electrophoresis
1) Acrylamide
2) Laemmli sample buffer – to prepare protein samples for SDS-PAGE
3) Running buffer
Acts as an electrolyte for running the gel (counteracts changes in pH arising from
electrolysis of water and has enough electrolytes to conduct electricity to run gel
in a short time. It is sold as a 10x concentrate.
4) Stacking gel
5) Resolving gel
6) Ethanol
7) SDS (10%)
8) Staining solution
9) Destaining solution and reservoir

Wear gloves at all times during reagent preparation and casting gel.
Acrylamide is a neurotoxin; precast gel in gel cassette is stored in
sodium azide, a bacterial inhibitor and toxic to humans

69
 Reagents for vertical gel electrophoresis
Stock solutions
Solution A: Acrylamide
• 30 g acrylamide/0.8 g Bis to 100 ml with Super Q water, sterilize by filtration
through 0.2 μm membrane filter
Solution B:
1.5 M Tris, pH 8.8 = 36.3 g Tris in 100 ml water. Adjust pH to 8.8 and adjust volume
to 200ml.
Solution C:
• 0.5 M Tris, pH 6.8 = 6 g Tris in 40 ml water. Adjust pH to 6.8 and adjust volume to
100 ml.
Solution D: ammonium persulfate (APS)
• 10% APS = 0.1 g in 1 ml water
Solution E: 10% SDS
3% Stacking gel
6.3 ml water/2.5 ml soln C/0.1 ml 10% SDS/1.2 ml soln A/10 µl TEMED/100 µl APS,
(lower pH of 6.8)
5X Tray or Running Buffer/liter (Tris-Glycine-SDS)
• 15 g Tris/72 g glycine/5 g SDS. Dilute 1:5 for upper tray and 1:10 for lower tray. This
is equivalent to 25 mM Tris /192 mM Glycine/0.1 % SDS (Higher pH of 8.8)

70
 Reagents for vertical gel electrophoresis
• 2x Laemmli Bio-Rad sample buffer – to prepare protein samples for
SDS-PAGE. Based on Tris-glycine-SDS running buffer. Ensures optimal
band resolution
Contains:
• 2.5 mM Tris-HCl, pH 6.8
• 25 % glycerol to make sample denser so it can sink to bottom of well
• 2 % SDS
• 0.01 % Bromophenol Blue
To use: (50:50 with sample)
• Dilute 1 part sample with 1 part Laemmli sample buffer.
• Add reducing agent to Laemmli : ß -mercaptoethanol (β-ME) or
dithiothreitol (DTT)
The reducing agent breaks disulfide linkages in proteins
Add 50 µl of β-ME per 950 µl of sample
buffer to a final concentration of 5 % ß –ME equivalent to 710 mM.
Alternatively, use DTT at a final concentration of 350 mM (54 mg/ml).
Add 1 mM EDTA to disperse cells

71
 10 % Resolving gel 3 % Stacking gel
pH 8.8 pH 6.8
45 ml 12 ml
Solution A (ml) 15 3.6
Solution B (ml) 7.5 -
Solution C (ml) - 2.5
10% SDS (ml) 0.45 0.1
(Solution E)
TEMED (μl) 20 1
APS (μl) 200 19
Water (ml) 21.83 5.78
72
 Resolving gel composition
29 ml 45 ml
Component 5% 7.5% 10% 5% 7.5% 10%

Soln. A (ml) 5 7.5 10 7.5 11.25 15

Soln. B (ml) 7.5 7.5 7.5 7.5 7.5 7.5

SDS (ml) 0.3 0.3 0.3 0.45 0.45 0.45

TEMED (μl) 15 15 15 20 20 20

APS (μl) 150 150 150 200 200 200

Water 16.1 13.6 11.7 24.15 20.4 17.55

73
 Preparation of PAGE gels
• To obtain optimal resolution of proteins, two gel systems
are used: a “stacking” gel which is cast over the top of the
“resolving” gel
• Stacking gel: has lower concentration of acrylamide (e.g.,
3% for larger pore size), lower pH of 6.8 and different ionic
content
• These properties of the stacking gel allow proteins in a
loaded sample to be concentrated into a tight band during
the first few minutes of electrophoresis before entering the
resolving portion of a gel
• If using gradient gel, the use of a stacking gel is not
necessary as the gradient, with its continually decreasing
pore size performs this function
• To make stacking gel: Mix all solutions, add TEMED last
• To make resolving gel: Mix all solutions, add TEMED last

74
Vertical gel
electrophoresis

75
 Assembly of apparatus and casting a gel
• The gel is cast between two glass plates, separated by
spacers, typically less than 2mm thick
To assemble
• Place the spacers on the long plate (spacer plate). Place short
notched plate on top of spacer plate so spacer is between the
two glass plates. Keep the short plate in front. Top side of
spacer plate is marked by manufacturer
• Slide both plates into the casting frame and secure it by
locking the pressure clamps. To tighten and lock, move clamps
through 90o
• Insure that both plates reach the bottom and align them
perfectly. A gasket in the casting frame makes a seal. Check
that the bottom is flat, i.e., glass plates do not protrude at the
base of casting frame. Place casting frame on the casting
stand
•

76
77
 Assembly of apparatus and casting a gel

• Place comb in between glass plates. Make a mark on

the right of glass plate, about 1 cm down the comb.
Check for leakages by spraying water into the space
between the glass plate. If no leakages, blot the
water with filter paper. Remove the comb and cast
the resolving gel

Precast gel in clamping frame

and electrode assembly 78
 Assembly of apparatus and casting a gel
• To cast the resolving gel, pour gel mix between the two
glass plates to the black mark you made outside the glass,
avoiding bubbles. Gel must be poured right away after
adding TEMED
• Remove any bubbles by adding a layer of isopropanol on
top of the gel way up to the top.
• Wait for gel to polymerize. Blot out the isopropanol using a
strip of filter paper or pour it off. After drying the
isopropanol, cast the stacking gel immediately after adding
TEMED
• Pour stacking gel on top of resolving gel to fill up the plates.
Be careful not to introduce any air bubble. Place comb in
the stacking gel which will create wells for samples. Wait
for gel to polymerize. Remove the comb and place unit in a
gel box. 79
 Using a precast gel
Use a gel casting clamping frame. Side arm
Place buffer dam in the gel casting
frame having two sides and gaskets Clamping frame
to hold plates in place. This frame
can run 2 gels, one on each side, or
run one gel with the other side
occupied by the buffer dam.
Place buffer dam in frame and
clamp side arms to secure the plate.
Remove gel cassette from storage
pouch and remove the protective
tape from the bottom to prevent
insulation of the gel cassette.
This cassette has a comb already in
place, and when placed in the
frame must face the inside of the
gel compartment.
80
 Electrophoresis
• Remove the glass plates from the stand and clamp and place
in gel box/electrophoresis tank
• Add buffer to outer and inner reservoir. Fill the outer
compartment with buffer to the mark indicated on gel box.
• Fill the inner compartment with buffer to the mark on gel box.
The level of buffer on the inner compartment will be higher
than that of the outer. This is used to ensure that there is no
leakage, since as if there is, the level of buffer in the inner will
drop down. Remove the comb gently and straight up
• Add more buffer to cover the inner glass plate to flood the
wells with the buffer
• Load gel: prepare samples with the loading buffer (Laemmli
loading buffer) and, if required, denature by heating a sample
and adding denaturing agents.
81
 Electrophoresis
• Use micropipette tips with slender ends to load sample wells.
Load 1st and 2nd wells with molecular marker and positive
control. Load sample wells
• After loading attach lid to gel box, red to red and black to
black. Connect to power pack, red to red and black to black.
• Turn on power supply and choose either constant current or
constant voltage. Press the run icon on power supply and
observe the development of a line of bubbles in the tank- a
sign that electrophoresis has began.
• The bubbles come from electrolysis of water and so the
bubbles are hydrogen gas.

82
Electrophoresis
About 20 min into
running of the gel, you
want to see sample
compressed into a
narrow line caused by
the sample moving
through the fairly low
percentage of the
stacking gel into the
high percentage and
stiffer resolving gel
causing the sample to
compress into a tight
line.

83
 Electrophoresis
• Run electrophoresis till the dye front reaches
appreciable length down the gel, before
bromophenol blue reaches end
• When the electric field is turned off, the proteins
stop moving. The gel matrix holds the proteins at
their final positions long enough for them to be
stained to make them visible.
• The gel material can also withstand high voltage
gradients, feasible for various staining and destaining
procedures, and can be cut to extract separated
fractions or dried for autoradiography and
permanent recording
84
 Polyacrylamide gels

• Separation is achieved by (1) net charge

carried by the protein and (2) sieving effect of
the gel matrix.
• Proteins segregate into discrete zones,
corresponding to their individual gel-mediated
mobilities
• Lanes of protein bands are created

85
 Polyacrylamide gels

• Separation by charge: in native PAGE, migration occurs

because most proteins carry a net negative charge at
slightly basic pH. The higher the negative charge, the
faster the protein will migrate
• Separation by sieving effect of the gel: the frictional
force created by the pore sizes of the gel matrix creates
a sieving effect, retarding the movement of proteins
according to their size.
• Small proteins encounter small frictional force while
large proteins face larger frictional force and are
retarded the most

86
 Gradient gels
• Gradient gels can be applied to protein and nucleic acid
separation
• Main features of a gradient gel
• Low percentage of acrylamide at the top (beginning of
sample path) and high percent-acrylamide at the
bottom (end) unlike homogeneous gels
• Pore size decreases linearly as proteins move down the
gel, hence migration rates decreases down the gel
• Are required for separation of both large and small
molecular weight proteins on a single gel slab
• Used for a quick estimate of the range of protein sizes
in a mixture permitting proper selection of a single
concentration gel
• Resolution is much better in homogeneous gel

87
 Gradient gels

• Another form of gradient gel

• Increasing concentration of denaturing reagent
(e.g., SDS for protein; urea and formamide for
DNA).
• Denaturing gradient gel electrophoresis (DGGE) is
used to resolve PCR amplicons up to a single base
difference, and separate out DNA on the basis of
variations in GC content.
• Find applications in elucidating molecular
diversity among microbial organism
88