Polyacrylamide gel electrophoresis

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Picture of an SDS-PAGE. The molecular markers (ladder) are in the left lane

Polyacrylamide gel electrophoresis (PAGE), describes a technique widely used

in biochemistry, forensics, genetics, molecular biologyand biotechnology to separate
biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic
mobility. Mobility is a function of the length, conformation and charge of the molecule.
As with all forms of gel electrophoresis, molecules may be run in their native state, preserving
the molecules' higher-order structure, or a chemical denaturant may be added to remove this
structure and turn the molecule into an unstructured linear chain whose mobility depends only
on its length and mass-to-charge ratio. For nucleic acids, urea is the most commonly used
denaturant. For proteins,sodium dodecyl sulfate (SDS) is an anionic detergent applied to protein
samples to linearize proteins and to impart a negative charge to linearized proteins. This
procedure is called SDS-PAGE. In most proteins, the binding of SDS to the polypeptide chain
imparts an even distribution of charge per unit mass, thereby resulting in a fractionation by
approximate size during electrophoresis. Proteins that have a greater hydrophobic content, for
instance many membrane proteins, and those that interact with surfactants in their native
environment, are intrinsically harder to treat accurately using this method, due to the greater
variability in the ratio of bound SDS.[1]
Contents
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Procedure[edit]
Sample preparation[edit]
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. 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.

Reduction of a typical disulfide bondby DTT via two sequential thiol-disulfide exchange reactions.

The sample to analyze is optionally mixed with a chemical denaturant if so desired,

usually SDS for proteins or urea for nucleic acids. SDS is an
anionic detergent that denatures secondary and nondisulfidelinked 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.[2][3][4][5]
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.

Preparing acrylamide gels[edit]

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 surface
smooth. [6] A source of free radicals and a stabilizer, such as ammonium
persulfate and TEMED are added to initiate polymerization.[7] 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 in 35. 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.

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.

Electrophoresis[edit]
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
different.[8][9] [10]

An electric field is applied across the gel, causing the negatively charged proteins or nucleic
acids to migrate across the gel away from the negative electrode (which is the cathode being
that this is an electrolytic rather than galvanic cell) and towards the positive electrode (the
anode). Depending on their size, each biomolecule moves differently through the gel matrix:
small molecules more easily fit through the pores in the gel, while larger ones have more
difficulty. 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. 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.

Further processing[edit]

Two SDS-PAGE-gels after a completed run

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.
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.

Chemical ingredients and their roles[edit]

Polyacrylamide gel (PAG) had been known as a potential embedding medium for sectioning
tissues as early as 1964, and two independent groups employed PAG in electrophoresis in
1959.[11][12] It possesses several electrophoretically desirable features that make it a versatile
medium. It is a synthetic, thermo-stable, transparent, strong, chemically relatively inert gel, and
can be prepared with a wide range of average pore sizes.[13] The pore size of a gel is determined
by two factors, the total amount of acrylamide present (%T) (T = Total concentration of
acrylamide and bisacrylamide monomer) and the amount of cross-linker (%C) (C =
bisacrylamide concentration). Pore size decreases with increasing %T; with cross-linking, 5%C
gives the smallest pore size. Any increase or decrease in %C from 5% increases the pore size,
as pore size with respect to %C is a parabolic function with vertex as 5%C. This appears to be
because of non-homogeneous bundling of polymer strands within the gel. This gel material can
also withstand highvoltage gradients, is amenable to various staining and destaining
procedures, and can be digested to extract separated fractions or dried for autoradiography and
permanent recording.

Components[edit]

Chemical buffer Stabilizes the pH value to the desired value within the gel itself and in
the electrophoresis buffer. The choice of buffer also affects the electrophoretic mobility of
the buffer counterions and thereby the resolution of the gel. 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.

Counterion 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 SDSPAGE cathode buffers, but may be included in the gel itself, where it migrates ahead of the
protein. In applications such as DISC SDS-PAGE the pH values within the gel may vary to
change the average charge of the counterions during the run to improve resolution. Popular
counterions are glycine and tricine. Glycine has been used as the source of trailing ion or
slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility
can be set at a value below that of the slowest known proteins of net negative charge in the
pH range. The minimum pH of this range is approximately 8.0.

Acrylamide (C3H5NO; mW: 71.08). 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 Vinyladdition
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 andhydrolysis.

Bisacrylamide (N,N-Methylenebisacrylamide) (C7H10N2O2; mW:

154.17). 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.

Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S; mW: 288.38). (only used in denaturing
protein gels) SDS is a strong detergent agent used to denature native proteins to unfolded,
individual polypeptides. When a protein mixture is heated to 100 C in presence of SDS,
the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant
weight ratio of 1.4 g SDS/g of polypeptide. In this process, the intrinsic charges of
polypeptides become negligible when compared to the negative charges contributed by
SDS. Thus polypeptides after treatment become rod-like structures possessing a uniform
charge density, that is same net negative charge per unit weight. The electrophoretic
mobilities of these proteins is a linear function of the logarithms of their molecular weights.
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; mW: 60.06). Urea is a chaotropic agent that increases the entropy of
the system by interfering with intramolecular interactions mediated by noncovalentforces 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 macromolecules,

Ammonium persulfate (APS) (N2H8S2O8; mW: 228.2). 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; mW: 116.21). 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 10 mM.

Chemicals for processing and visualization[edit]

PAGE of rotavirus proteins stained with Coomassie blue

The following chemicals and procedures are used for processing of the gel and the protein
samples visualized in it:

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 nonionic and non-reactive towards proteins to avoid interfering with electrophoresis.
Common additives are glycerol and sucrose.

Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97). CBB is the most
popular protein stain. It is an anionic dye, which non-specifically binds to proteins. The
structure of CBB is predominantly non-polar, and it is usually used in methanolic
solution acidified with acetic acid. 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.

Ethidium bromide (EtBr) 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. The exact
chemical mechanism by which this happens is still largely unknown.[14] Silver staining
was introduced by Kerenyi and Gallyas as a sensitive procedure to detect trace
amounts of proteins in gels.[15] The technique has been extended to the study of other
biological macromolecules that have been separated in a variety of supports.[16] Many
variables can influence the colour intensity and every protein has its own staining
characteristics; clean glassware, pure reagents and water of highest purity are the key
points to successful staining.[17] Silver staining was developed in the 14th century for
colouring the surface of glass. It has been used extensively for this purpose since the
16th century. The colour produced by the early silver stains ranged between light yellow
and an orange-red. Camillo Golgi perfected the silver staining for the study of
the nervous system. Golgi's method stains a limited number of cells at random in their
entirety.[18]