• TYPES OF ELECTROPHORESIS
• GEL ELECTROPHORESIS
• 1-D GEL ELECTROPHORESIS
• POLYACRYLAMIDE GEL
• STACKING AND RESOLVING GELS
• 2-D GEL ELECTROPHORESIS
• AN OVERVIEW OF SDS-PAGE
• ADVANTAGES & DISADVANTAGES
• APPLICATIONS OF SDS-PAGE
Electrophoresis is the process of moving charged molecules in solution by applying an
electric ﬁeld across the mixture. Because molecules in an electric ﬁeld move with a speed
dependent on their charge, shape, and size, electrophoresis has been extensively developed
for molecular separations.
• Purpose for carrying out electrophoresis:
1. To determine the number, amount and mobility of components in a given sample or to
2. To obtain information about the electrical double layers surrounding the particles.
3. Determination of molecular weight of proteins and DNA sequencing.
• Any charged ion or molecule migrates when placed in an electric field. The rate of migration
depend upon its net charge, size, shape and the applied electric current.
• It can be represented by following equation:
v = velocity of migration of the molecule
E = electric field (in volts/cm)
q = net electric charge on the molecule.
F = frictional co-efficient (depends upon the mass and shape of the molecule).
• The movement of charged particle in an electric field is expressed in terms of electrophoretic
mobility denoted by ‘µ’, where 𝜇 = 𝑣 𝐸 or 𝜇 = 𝑞 𝐹
• For molecules with similar conformation, 𝐹 varies with size but not with shape. Thus
electrophoretic mobility (µ) of a molecule is directly proportional to the charge density
6. GEL ELECTROPHORESIS
• It is a technique used for the separation of Deoxyribonucleic acid, Ribonucleic acid or protein
molecules according to their size and electrical charge using an electric current applied to a gel
• What is a gel?
Gel is a cross linked polymer whose composition and porosity is chosen based on the specific
weight and porosity of the target molecules.
• Types of gels used: Polyacrylamide gel, Agarose gel.
• Types of gel electrophoresis:
One dimensional (1-D) gel
SDS-PAGE Native PAGE
Two dimensional (2-D) gel
Separates proteins by
isoelectric point in the
first dimension and by
mass in the second
7. 1-D GEL ELECTROPHORESIS
The samples are treated with SDS (sodium dodecyl sulfate), an anionic detergent which
denatures the protein by breaking the disulfide bonds and gives negative charge to each
protein in proportion to its mass.
Without SDS, different proteins with similar molecular weights would migrate differently due
to differences in folding, as differences in folding patterns would cause some proteins to
better fit through the gel matrix than others. SDS linearizes the proteins so that they may be
separated strictly by molecular weight.
The SDS binds to the protein in a ratio of approximately 1.4 g SDS per 1.0 g giving an
approximately uniform mass : charge ratio for most proteins, so that the distance of migration
through the gel can be assumed to be directly related to only the size of the protein.
Proteins may be further treated with reducing agent, such as dithiothreitol (DTT) or TRP
(Tributyl phosphine) to break any reformed disulfide bonds and then alkalated with
iodoacetamide to prevent reformation of disulfide bonds.
A tracking dye like bromophenol blue may be added to the protein solution to track the
progress of the protein solution through the gel during the electrophoretic run.
8. POLYACRYLAMIDE GEL
• The gel used for SDS-PAGE is made out of acrylamide which form cross-linked polymers of
• Standard gels are typically composed of two layers, one top-most layer called the stacking gel
and a lower layer called separating or resolving gel.
• The stacking layer contains a low percentage of acylamide and has low pH , while the acrylamide
concentration of the separating gel varies according to the samples to be run and has higher pH.
• The difference in pH and acrylamide concentration at the stacking and separating gel provides
better resolution and sharper bands in the separating gel.
• This gel material can also withstand high voltage gradients, is amenable to various staining and
destaining procedures, and can be digested to extract separated fractions or dried for
autoradiography and permanent recording.
9. STACKING & RESOLVING GELS
• Stacking gel:
The stacking gel is a large pore PAG (4%T). This gel is prepared with Tris/HCl buffer pH 6.8 of
about 2.0 pH units lower than that of electrophoresis buffer (Tris/Glycine).
This gel is cast over the resolving gel. The height of the stacking gel region is always
maintained more than double the height and th volume of the sample to be applied.
• Resolving gel:
The resolving gel is a small pore polyacrylamide gel (3 -30% acrylamide monomer) typically
made using a pH 8.8 Tris/HCl buffer.
In the resolving gel, macromolecules separate according to their size. Resolving gels have an
optimal range of separation that is based on the percent of monomer present in the
10. 2-D GEL ELECTROPHORESIS
• Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel
electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two
properties in two dimensions on 2D gels.
• 2-DE was first independently introduced by O'Farrell and Klose in 1975.
• 2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a
second property in a direction 90 degrees from the first. In 1-D electrophoresis, proteins (or other
molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane
but that the molecules are spread out across a 2-D gel. Because it is unlikely that two molecules
will be similar in two distinct properties, molecules are more effectively separated in 2-D
electrophoresis than in 1-D electrophoresis.
• The two dimensions that proteins are separated into using this technique can be isoelectric point,
protein complex mass in the native state, and protein mass.
• Common techniques of 2-DE:
11. AN OVERVIEW OF SDS-PAGE
• SDS-PAGE is a technique used by many researchers to separate mixtures of proteins by their
• Sodium dodecyl sulfate (SDS) is a detergent that breaks up the interactions between proteins.
• SDS is an anionic detergent that disrupts secondary and non-disulfide-linked tertiary structures
and additionally applies a negative charge to each protein in proportion to its mass.
Fig. 1. An overview of SDS-PAGE
12. AN OVERVIEW OF SDS-PAGE
• PRINCIPAL COMPONENTS OF SDS-PAGE:
The components of an SDS PAGE gel electrophoresis system are the following:
1. A Slab holder for vertical or horizontal gels (thin, flat sheets of many individual lanes)
2. Polyacrylamide or agarose gels (cm x cm x mm); these are poured for each analysis
3. Gel is amended with SDS to dissociate & charge proteins.
4. High voltage power supply (0.1-6 kV)
5. A detection technique (dye staining, fluorescence, or autoradiography to image separated
SDS-PAGE stands for Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis. Sodium-
Dodecyl Sulfate, (“SDS”), is an anionic detergent. It is composed of a hydrophilic group with a
net negative charge and a long hydrophobic chain with neutral charge.
• SAMPLE PREPARATION:
Samples may be any material containing proteins or nucleic acids. These may be biologically
derived, for example from prokaryotic or eukaryotic cells, tissues, viruses, environmental
samples, or purified proteins.
13. AN OVERVIEW OF SDS-PAGE
In the case of solid tissues or cells, these are often first broken down mechanically using a
blender (for larger sample volumes), using a homogenizer (smaller volumes), by sonicator or
by using cycling of high pressure, and a combination of biochemical and mechanical
techniques – including various types of filtration and centrifugation – may be used to separate
different cell compartments and organelles prior to electrophoresis. Synthetic biomolecules
such as oligonucleotides may also be used as analytes.
The sample to analyze is optionally mixed with a chemical denaturant if so desired, usually SDS
for proteins or urea for nucleic acids.
SDS denatures secondary and non–disulfide–linked tertiary structures, and additionally applies
a negative charge to each protein in proportion to its mass. Urea breaks the hydrogen bonds
between the base pairs of the nucleic acid, causing the constituent strands to separate.
Heating the samples to at least 60 °C further promotes denaturation.
In addition to SDS, proteins may optionally be briefly heated to near boiling in the presence of
a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-
mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages,
thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein
structure (oligomeric subunits). This is known as reducing SDS-PAGE.
A tracking dye may be added to the solution. This typically has a higher electrophoretic
mobility than the analytes to allow the experimenter to track the progress of the solution
through the gel during the electrophoretic run.
14. AN OVERVIEW OF SDS-PAGE
Fig. 2. Sample preparation for SDS-PAGE electrophoresis
• PREPARATION OF ACRYLAMIDE GELS:
The gels typically consist of acrylamide, bisacrylamide, the optional denaturant (SDS or urea),
and a buffer with an adjusted pH. The solution may be degassed under a vacuum to prevent
the formation of air bubbles during polymerization. Alternatively, butanol may be added to
the resolving gel (for proteins) after it is poured, as butanol removes bubbles and makes the
A source of free radicals and a stabilizer, such as ammonium persulfate and TEMED are added
to initiate polymerization. The polymerization reaction creates a gel because of the added
bisacrylamide, which can form cross-links between two acrylamide molecules. The ratio of
bisacrylamide to acrylamide can be varied for special purposes, but is generally about 1 part
15. AN OVERVIEW OF SDS-PAGE
The acrylamide concentration of the gel can also be varied, generally in the range from 5% to
25%. Lower percentage gels are better for resolving very high molecular weight molecules,
while much higher percentages are needed to resolve smaller proteins.
Fig. 3. Mixture of acrylamide, bisacrylamide, the optional denaturant (SDS or urea), and a buffer with an adjusted pH,
followed by addition of ammonium persulfate and TEMED to initiate polymerization and subsequent stacking.
Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at
the top to create the sample wells. After the gel is polymerized the comb can be removed and
the gel is ready for electrophoresis.
16. AN OVERVIEW OF SDS-PAGE
Fig. 4. Polymerization of acrylamide gel using a gel caster
Various buffer systems are used in PAGE depending on the nature of the sample and the
experimental objective. The buffers used at the anode and cathode may be the same or
The gel is run usually for a few hours, though this depends on the voltage applied across the
gel; migration occurs more quickly at higher voltages, but these results are typically less
accurate than at those at lower voltages. After the set amount of time, the biomolecules have
migrated different distances based on their size.
17. AN OVERVIEW OF SDS-PAGE
Smaller biomolecules travel farther down the gel, while larger ones remain closer to the point
of origin. Biomolecules may therefore be separated roughly according to size, which depends
mainly on molecular weight under denaturing conditions, but also depends on higher-order
conformation under native conditions. However, certain glycoproteins behave anomalously
on SDS gels.
Fig. 5. Preparation of anode and cathode buffers
18. AN OVERVIEW OF SDS-PAGE
Fig. 6. Electrophoresis and subsequent formation of separate bands on the gel cassette
• FURTHER PROCESSING:
Following electrophoresis, the gel may be stained (for proteins, most commonly with
Coomassie Brilliant Blue R-250; for nucleic acids, ethidium bromide; or for either, silver stain),
allowing visualization of the separated proteins, or processed further (e.g. Western blot). After
staining, different species biomolecules appear as distinct bands within the gel. It is common
to run molecular weight size markers of known molecular weight in a separate lane in the gel
to calibrate the gel and determine the approximate molecular mass of unknown biomolecules
by comparing the distance traveled relative to the marker.
19. AN OVERVIEW OF SDS-PAGE
For proteins, SDS-PAGE is usually the first choice as an assay of purity due to its reliability and
ease. The presence of SDS and the denaturing step make proteins separate, approximately
based on size, but aberrant migration of some proteins may occur. Different proteins may
also stain differently, which interferes with quantification by staining. PAGE may also be used
as a preparative technique for the purification of proteins. For example, quantitative
preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE) is a method
for separating native metalloproteins in complex biological matrices.
Fig. 7. Two SDS-PAGE-gels after a completed run
20. AN OVERVIEW OF SDS-PAGE
• Components of SDS-PAGE:
Chemical buffer: Stabilizes the pH value to the desired value within the gel itself and in the
electrophoresis buffer. The buffer should also be unreactive and not modify or react with most
proteins. Different buffers may be used as cathode and anode buffers, respectively, depending
on the application. Multiple pH values may be used within a single gel, for example in DISC
electrophoresis. Common buffers in PAGE include Tris, Bis-Tris, or imidazole.
Counter-ion: Balance the intrinsic charge of the buffer ion and also affect the electric field
strength during electrophoresis. Highly charged and mobile ions are often avoided in SDS-
PAGE cathode buffers, but may be included in the gel itself, where it migrates ahead of the
Acrylamide (C3H5NO): When dissolved in water, slow, spontaneous autopolymerization of
acrylamide takes place, joining molecules together by head on tail fashion to form long single-
chain polymers. The presence of a free radical-generating system greatly accelerates
polymerization. This kind of reaction is known as Vinyl addition polymerisation. A solution of
these polymer chains becomes viscous but does not form a gel, because the chains simply
slide over one another. Gel formation requires linking various chains together. Acrylamide is a
neurotoxin. It is also essential to store acrylamide in a cool dark and dry place to reduce
autopolymerisation and hydrolysis.
Bisacrylamide (N,N′-Methylenebisacrylamide) (C7H10N2O2): Bisacrylamide is the most frequently
used cross linking agent for polyacrylamide gels. Chemically it can be thought of as two
acrylamide molecules coupled head to head at their non-reactive ends. Bisacrylamide can
crosslink two polyacrylamide chains to one another, thereby resulting in a gel.
21. AN OVERVIEW OF SDS-PAGE
Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S): SDS is a strong detergent agent used to denature
native proteins to unfolded, individual polypeptides. Without SDS, different proteins with
similar molecular weights would migrate differently due to differences in mass-charge ratio, as
each protein has an isoelectric point and molecular weight particular to its primary structure.
This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the
protein, giving a near uniform negative charge along the length of the polypeptide.
Urea (CO(NH2)2): Urea is a chaotropic agent that increases the entropy of the system by
interfering with intramolecular interactions mediated by non-covalent forces such as hydrogen
bonds and van der Waals forces. Macromolecular structure is dependent on the net effect of
these forces, therefore it follows that an increase in chaotropic solutes denatures
Ammonium persulfate (APS) (N2H8S2O8): APS is a source of free radicals and is often used as an
initiator for gel formation. An alternative source of free radicals is riboflavin, which generated
free radicals in a photochemical reaction.
TEMED (N, N, N′, N′-tetramethylethylenediamine) (C6H16N2): TEMED stabilizes free radicals and
improves polymerization. The rate of polymerisation and the properties of the resulting gel
depend on the concentrations of free radicals. Increasing the amount of free radicals results in
a decrease in the average polymer chain length, an increase in gel turbidity and a decrease in
gel elasticity. Decreasing the amount shows the reverse effect. The lowest catalytic
concentrations that allow polymerisation in a reasonable period of time should be used. APS
and TEMED are typically used at approximately equimolar concentrations in the range of 1 to
22. AN OVERVIEW OF SDS-PAGE
• Chemicals used for processing and visualization:
Tracking dye: As proteins and nucleic acids are mostly colorless, their progress through the
gel during electrophoresis cannot be easily followed. Anionic dyes of a known electrophoretic
mobility are therefore usually included in the PAGE sample buffer. A very common tracking
dye is Bromophenol blue (BPB, 3',3",5',5" tetrabromophenolsulfonphthalein). This dye is
coloured at alkali and neutral pH and is a small negatively charged molecule that moves
towards the anode. Being a highly mobile molecule it moves ahead of most proteins. As it
reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can
weakly bind to some proteins and impart a blue colour. Other common tracking dyes are
xylene cyanol, which has lower mobility, and Orange G, which has a higher mobility.
Loading aids: Most PAGE systems are loaded from the top into wells within the gel. To ensure
that the sample sinks to the bottom of the gel, sample buffer is supplemented with additives
that increase the density of the sample. These additives should be non-ionic and non-reactive
towards proteins to avoid interfering with electrophoresis. Common additives are glycerol
Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2): CBB is the most popular protein stain. It
is an anionic dye, which non-specifically binds to proteins. Proteins in the gel are fixed by
acetic acid and simultaneously stained. The excess dye incorporated into the gel can be
removed by destaining with the same solution without the dye. The proteins are detected as
blue bands on a clear background. As SDS is also anionic, it may interfere with staining
process. Therefore, large volume of staining solution is recommended, at least ten times the
volume of the gel.
23. AN OVERVIEW OF SDS-PAGE
Ethidium bromide (EtBr): It is the traditionally
most popular nucleic acid stain.
Silver staining: Silver staining is used when
more sensitive method for detection is
needed, as classical Coomassie Brilliant Blue
staining can usually detect a 50 ng protein
band, Silver staining increases the sensitivity
typically 50 times. Silver staining was
introduced by Kerenyi and Gallyas as a
sensitive procedure to detect trace amounts
of proteins in gels.
Western Blotting: It is a process by which
proteins separated in the acrylamide gel are
electrophoretically transferred to a stable,
manipulable membrane such as a
nitrocellulose, nylon, or PVDF membrane. It
is then possible to apply immunochemical
techniques to visualise the transferred
proteins, as well as accurately identify
relative increases or decreases of the protein
Fig. 8. Coomassie staining and Silver staining
24. AN OVERVIEW OF SDS-PAGE
Ethidium bromide (EtBr): It is the traditionally most popular nucleic acid stain.
Silver staining: Silver staining is used when more sensitive method for detection is needed, as
classical Coomassie Brilliant Blue staining can usually detect a 50 ng protein band, Silver
staining increases the sensitivity typically 50 times. Silver staining was introduced by Kerenyi
and Gallyas as a sensitive procedure to detect trace amounts of proteins in gels.
Western Blotting: It is a process by which proteins separated in the acrylamide gel are
electrophoretically transferred to a stable, manipulable membrane such as a nitrocellulose,
nylon, or PVDF membrane. It is then possible to apply immunochemical techniques to
visualise the transferred proteins, as well as accurately identify relative increases or decreases
of the protein of interest.
25. ADVANTAGES & DISADVANTAGES
1. Migration of the molecules is proportional to their molecular weights.
2. Highly sensitive test, separates molecules that have even a 2% difference in mass.
3. Requires very small amounts of samples.
4. A stable chemically cross-linked gel is used.
1. Poor band resolution due to high alkaline operating pH.
2. Acrylamide gel is a potent neurotoxin chemical.
3. Gel preparation is difficult and takes a long time.
4. Very costly.
26. APPLICATIONS OF SDS-PAGE
SDS-PAGE is used mainly for the following purposes:
1. Measuring molecular weight.
2. Peptide mapping.
3. Estimation of protein size.
4. Determination of protein subunits or aggregation structures.
5. Estimation of protein purity.
6. Protein quantitation.
7. Monitoring protein integrity.
8. Comparison of the polypeptide composition of different samples.
9. Analysis of the number and size of polypeptide subunits.
10. Post-electrophoresis applications, such as Western blotting.
11. Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid.
12. Pouring and Running a Protein Gel by reusing Commercial Cassettes.
13. Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes.
14. Detection of Protein Ubiquitination.
15. SDS-PAGE/Immunoblot Detection of Aβ Multimers in Human Cortical Tissue Homogenates
using Antigen-Epitope Retrieval.
27. APPLICATIONS OF SDS-PAGE
• Measuring Molecular Weight with SDS-PAGE:
The mobility (Rf) of a molecule in gel electrophoresis is determined by its free solution
mobility, Y0 (= mobility in a gel of zero %) and the sieving action of the gel matrix. In
denaturing protein electrophoresis, the addition of SDS to the electrophoresis buffer
uniformly coats the proteins with negative charges, equalizing the charge to mass ratio for
all proteins, thus making Y0 he same for all species. In this case, relative mobilities are
determined solely by the sieving action of the gel. This sieving action is proportional to the
molecular weight (MW) of the particular protein. Theoretical treatments suggest that the
logarithm of Rf should vary with MW, but most users use an empirical plot of the logarithm
of MW vs Rf for several standards of known molecular weights to determine the molecular
weights of unknowns.
In practice the proportionality of log(MW) vs Rf holds true for most proteins, provided they
are fully denatured, and provided the gel percentage has been chosen to match the
molecular weight range of the sample. In fact, the actual plot of log(MW) vs Rf is sigmoidal,
because at high MW, the sieving affect of the matrix is so large that molecules are unable to
penetrate the gel, while at low MW, the sieving effect is negligible, and proteins migrate
almost at their free mobility, which in SDS is independent of MW.
Given an appropriate selection of gel % and a protein which displays near-ideal behavior,
molecular weights can be determined to within 5 - 10%. Molecular weights of non-ideal
proteins can be determined by the use of Ferguson Plots, a technique which employs native
28. APPLICATIONS OF SDS-PAGE
Fig. 9. A graph of logMW vs. Rf. Although it is sigmoidal, it is nearly linear for a range of molecular weights depending
on the percentage monomer of the gel.
Determining Molecular Weight with Gradient SDS Gels: If a gradient of acrylamide
concentration is introduced into SDS PAGE, larger ranges of proteins may be analyzed on
the same gels, with greater resolution. The complexity of the relationship between migration
and molecular weight is dependant upon the shape of the gradient. The overall equation is
of the form log(MW) α log(P), where P is the concentration of acrylamide at the band
position. A graph of log(MW) vs log(P) is linear, and allows the determination of MW's from
a set of standard protein positions. For linear gradient gels, the percentage of acrylamide is
proportional to the position in the gel, so log(MW) will be proportional to log(band
position). Therefore, a graph of log(MW) vs log(Rf) for a set of standards will be linear, and Rf
values for unknowns can then be converted to MW values. On a 3 - 30% gradient gel, a
range of proteins which differ in MW by up to 100 fold can be resolved and MW's
29. APPLICATIONS OF SDS-PAGE
• Peptide Mapping:
Peptide mapping involves controlled cleavage of a pure protein with small amounts of a
pure protease to generate peptides of characteristic, reproducible sizes. These peptides can
be separated on PAGE to produce a "fingerprint" characteristic of the protein. Peptide
mapping can map cleavage sites in an unknown protein, or it can identify an unknown
protein based upon its fingerprint identity with a previously tested sample.
The polyacrylamide gel used can be either denaturing or non-denaturing, but SDS PAGE is
most often used because it gives molecular weight information about the peptides
produced. Small amounts of protease are used, so that minor variations in time and
temperature of incubations will not overly perturb the results.
Proteins for peptide mapping can be taken from bands sliced out of electrophoresis gels, or
purified by standard means. Protocols are provided for the mapping of a pure protein and a
protein in an acrylamide gel.
Analytical gels for peptide mapping: The choice of gel system for the analysis of peptide
mapping is dictated by the anticipated results. If a wide range of peptide sizes is anticipated,
a< gradient gel may be required. For peptides over 7kd, standard Tris Glycine SDS PAGE
gels will give superior results. Small peptides will require strongly denaturing fixatives to
avoid loss of signal during staining. For extremely small peptides, analysis on native PAGE
gels may be superior. In native protein PAGE, separation is based partly on charge to mass
ratio. This can enable the resolution of peptides that would run too close to the SDS/dye
front in SDS PAGE.
• SDS-PAGE is a major tool that has wide applications apart from analytical sciences.
• SDS-PAGE is a technique widely used in biochemistry, forensics, genetics, molecular biology and
biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according
to their electrophoretic mobility.
• This is an advancement in gel electrophoresis and the most widely used 1-D gel electrophoresis
• With the help of this, proteins can be separated from a mixture and individual proteins could be
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