SDS-PAGE

SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) is a

variant of polyacrylamide gel electrophoresis, an analytical method in
biochemistry for the separation of charged molecules in mixtures by their
molecular masses in an electric field. It uses sodium dodecyl sulfate (SDS)
molecules to help identify and isolate protein molecules.

SDS-PAGE is a discontinuous electrophoretic system developed by Ulrich K.

Laemmli which is commonly used as a method to separate proteins with
molecular masses between 5 and 250 KDa.[1] The publication describing it is the
most frequently cited paper by a single author, and the second most cited
overall.[2]

Contents Proteins of the erythrocyte

membrane separated by SDS-PAGE
Properties according to their molecular masses
Procedure
Gel production
Sample preparation
Electrophoresis
Gel staining
Analysis
Archiving
Molecular mass determination
Applications
Variants
Alternatives
History
References
External links

Properties
SDS-PAGE is an electrophoresis method that allows protein separation by mass.
The medium (also referred to as ′matrix′) is a polyacrylamide-based
discontinuous gel. In addition, SDS (sodium dodecyl sulfate) is used. About 1.4
grams of SDS bind to a gram of protein,[3][4][5] corresponding to one SDS
molecule per two amino acids. SDS acts as a surfactant, covering the proteins' Unfolding of a protein with SDS
intrinsic charge and conferring them very similar charge-to-mass ratios. The
intrinsic charges of the proteins are negligible in comparison to the SDS loading,
and the positive charges are also greatly reduced in the basic pH range of a separating gel. Upon application of a constant electric
field, the protein migrate towards the anode, each with a different speed, depending on its mass. This simple procedure allows
precise protein separation by mass.

SDS tends to form spherical micelles in aqueous solutions above a certain

concentration called the critical micellar concentration (CMC). Above the
critical micellar concentration of 7 to 10 millimolar in solutions, the SDS
simultaneously occurs as single molecules (monomer) and as micelles, below the
CMC SDS occurs only as monomers in aqueous solutions. At the critical
micellar concentration, a micelle consists of about 62 SDS molecules.[6] Unfolding of a protein with heat
However, only SDS monomers bind to proteins via hydrophobic interactions,
whereas the SDS micelles are anionic on the outside and do not adsorb any
protein.[3] SDS is amphipathic in nature, which allows it to unfold both polar and nonpolar sections of protein structure.[7] In
SDS concentrations above 0.1 millimolar, the unfolding of proteins begins,[3] and above 1 mM, most proteins are denatured.[3]
Due to the strong denaturing effect of SDS and the subsequent dissociation of protein complexes, quaternary structures can
generally not be determined with SDS. Exceptions are e.g. proteins that were previously stabilised by covalent cross-linking and
the SDS-resistant protein complexes, which are stable even in the presence of SDS (the latter, however, only at room
temperature). To denature the SDS-resistant complexes a high activation energy is required, which is achieved by heating. SDS
resistance is based on a metastability of the protein fold. Although the native, fully folded, SDS-resistant protein does not have
sufficient stability in the presence of SDS, the chemical equilibrium of denaturation at room temperature occurs slowly. Stable
protein complexes are characterised not only by SDS resistance but also by stability against proteases and an increased biological
half-life.[8]

Alternatively, polyacrylamide gel electrophoresis can also be performed with the cationic surfactants CTAB in a CTAB-
PAGE,[9][10][11] or 16-BAC in a BAC-PAGE.[12]

Procedure
The SDS-PAGE method is composed of gel preparation, sample preparation, electrophoresis, protein staining or western blotting
and analysis of the generated banding pattern.

Gel production
When using different buffers in the gel (discontinuous gel electrophoresis), the gels are made up to one day prior to
electrophoresis, so that the diffusion does not lead to a mixing of the buffers. The gel is produced by radical polymerisation in a
mold consisting of two sealed glass plates with spacers between the glass plates. In a typical mini-gel setting, the spacers have a
thickness of 0.75 mm or 1.5 mm, which determines the loading capacity of the gel. For pouring the gel solution, the plates are
usually clamped in a stand which temporarily seals the otherwise open underside of the glass plates with the two spacers. For the
gel solution, acrylamide is mixed as gel-former (usually 4% V/V in the stacking gel and 10-12 % in the separating gel),
methylenebisacrylamide as a cross-linker, stacking or separating gel buffer, water and SDS. By adding the catalyst TEMED and
the radical initiator ammonium persulfate (APS) the polymerisation is started. The solution is then poured between the glass
plates without creating bubbles. Depending on the amount of catalyst and radical starter and depending on the temperature, the
polymerisation lasts between a quarter of an hour and several hours. The lower gel (separating gel) is poured first and covered
with a few drops of a barely water-soluble alcohol (usually buffer-saturated butanol or isopropanol), which eliminates bubbles
from the meniscus and protects the gel solution of the radical scavenger oxygen. After the polymerisation of the separating gel,
the alcohol is discarded and the residual alcohol is removed with filter paper. After addition of APS and TEMED to the stacking
gel solution, it is poured on top of the solid separation gel. Afterwards, a suitable sample comb is inserted between the glass
plates without creating bubbles. The sample comb is carefully pulled out after polymerisation, leaving pockets for the sample
application. For later use of proteins for protein sequencing, the gels are often prepared the day before electrophoresis to reduce
reactions of unpolymerised acrylamide with cysteines in proteins.
By using a gradient mixer, gradient gels with a gradient of acrylamide (usually
from 4 to 12%) can be cast, which have a larger separation range of the
molecular masses.[13] Commercial gel systems (so-called pre-cast gels) usually
use the buffer substance Bis-tris methane with a pH value between 6.4 and 7.2
both in the stacking gel and in the separating gel.[14][15] These gels are delivered
cast and ready-to-use. Since they use only one buffer (continuous gel
electrophoresis) and have a nearly neutral pH, they can be stored for several
weeks. The more neutral pH slows the hydrolysis and thus the decomposition of
the polyacrylamide. Furthermore, there are fewer acrylamide-modified cysteines
in the proteins.[14] Due to the constant pH in collecting and separating gel there
is no stacking effect. Proteins in BisTris gels can not be stained with ruthenium
complexes.[16] This gel system has a comparatively large separation range,
which can be varied by using MES or MOPS in the running buffer.[14]

Sample preparation
Sample combs with different
During sample preparation, the sample buffer, and thus SDS, is added in excess numbers of pockets, each prong
to the proteins, and the sample is then heated to 95 °C for five minutes, or leaves a pocket in the gel when
alternatively 70 °C for ten minutes. Heating disrupts the secondary and tertiary pulled out
structures of the protein by disrupting hydrogen bonds and stretching the
molecules. Optionally, disulfide bridges can be cleaved by reduction. For this
purpose, reducing thiols such as β-mercaptoethanol (β-ME, 5% by volume),
dithiothreitol (DTT, 10 millimolar) or dithioerythritol (DTE, 10 millimolar) are
added to the sample buffer. After cooling to room temperature, each sample is
pipetted into its own well in the gel, which was previously immersed in
electrophoresis buffer in the electrophoresis apparatus.

In addition to the samples, a molecular-weight size marker is usually loaded onto

the gel. This consists of proteins of known sizes and thereby allows the
estimation (with an error of ± 10%) of the sizes of the proteins in the actual
samples, which migrate in parallel in different tracks of the gel.[17] The size Polymerised separating and stacking
gel before removing the sample
marker is often pipetted into the first or last pocket of a gel.
comb (white) between the spacers
(black), in the stacking gel are small
amounts of bromophenol blue for
Electrophoresis improved visibility, the separating gel
For separation, the denatured samples are loaded onto a gel of polyacrylamide, is unstained
which is placed in an electrophoresis buffer with suitable electrolytes.
Thereafter, a voltage (usually around 100 V, 10-20 V per cm gel length) is
applied, which causes a migration of negatively charged molecules through the
gel in the direction of the positively charged anode. The gel acts like a sieve.
Small proteins migrate relatively easily through the mesh of the gel, while larger
proteins are more likely to be retained and thereby migrate more slowly through
the gel, thereby allowing proteins to be separated by molecular size. The
Disulfide reduction by DTT
electrophoresis lasts between half an hour to several hours depending on the
voltage and length of gel used.
The fastest-migrating proteins (with a molecular weight of less than 5 KDa)
form the buffer front together with the anionic components of the electrophoresis
buffer, which also migrate through the gel. The area of the buffer front is made
visible by adding the comparatively small, anionic dye bromophenol blue to the
sample buffer. Due to the relatively small molecule size of bromophenol blue, it
migrates faster than proteins. By optical control of the migrating colored band,
the electrophoresis can be stopped before the dye and also the samples have
completely migrated through the gel and leave it.

The most commonly used method is the discontinuous SDS-PAGE. In this

method, the proteins migrate first into a collecting gel with neutral pH, in which
they are concentrated and then they migrate into a separating gel with basic pH,
in which the actual separation takes place. Stacking and separating gels differ by
different pore size (4-6 % T and 10-20 % T), ionic strength and pH values (pH
6.8 or pH 8.8). The electrolyte most frequently used is an SDS-containing Tris-
Electrophoresis chamber after a few
glycine-chloride buffer system. At neutral pH, glycine predominantly forms the minutes of electrophoresis. In the
zwitterionic form, at high pH the glycines lose positive charges and become first pocket a size marker was
predominantly anionic. In the collection gel, the smaller, negatively charged applied with bromophenol blue, in the
chloride ions migrate in front of the proteins (as leading ions) and the slightly other pockets, the samples were
larger, negatively and partially positively charged glycinate ions migrate behind added bromocresol green

the proteins (as initial trailing ions), whereas in the comparatively basic
separating gel both ions migrate in front of the proteins. The pH gradient
between the stacking and separation gel buffers leads to a stacking effect at the border of the stacking gel to the separation gel,
since the glycinate partially loses its slowing positive charges as the pH increases and then, as the former trailing ion, overtakes
the proteins and becomes a leading ion, which causes the bands of the different proteins (visible after a staining) to become
narrower and sharper - the stacking effect. For the separation of smaller proteins and peptides, the TRIS-Tricine buffer system of
Schägger and von Jagow is used due to the higher spread of the proteins in the range of 0.5 to 50 KDa.[18]

Gel staining
At the end of the electrophoretic separation, all proteins are sorted by size and can then be analyzed by other methods, e. g.
protein staining such as Coomassie staining (most common and easy to use),[19][20] silver staining (highest
sensitivity),[21][22][23][24][25][26] stains all staining, Amido black 10B staining,[20] Fast green FCF staining,[20] fluorescent stains
such as epicocconone stain[27] and SYPRO orange stain,[28] and immunological detection such as the Western Blot.[29][30] The
fluorescent dyes have a comparatively higher linearity between protein quantity and color intensity of about three orders of
magnitude above the detection limit, i. e. the amount of protein can be estimated by color intensity. When using the fluorescent
protein dye trichloroethanol, a subsequent protein staining is omitted if it was added to the gel solution and the gel was irradiated
with UV light after electrophoresis.[31][32]

In Coomassie Staining, Gel is Fixed in a 50% ethanol 10% glacial acetic acid solution for 1 hr. Then the solution is changed for
fresh one and after 1 to 12 hrs Gel is changed to a Staining solution (50% methanol, 10% Glacial acetic Acid, 0.1% Coomassie
Brilliant Blue) followed by destaining changing several times a destaining solution of 40% methanol, 10% glacial acetic acid.

Analysis
Protein staining in the gel creates a documentable banding pattern of the various proteins. Glycoproteins have differential levels
of glycosylations and adsorb SDS more unevenly at the glycosylations, resulting in broader and blurred bands.[33] Membrane
proteins, because of their transmembrane domain, are often composed of the more hydrophobic amino acids, have lower
solubility in aqueous solutions, tend to bind lipids, and tend to precipitate in
aqueous solutions due to hydrophobic effects when sufficient amounts of
detergent are not present. This precipitation manifests itself for membrane
proteins in a SDS-PAGE in "tailing" above the band of the transmembrane
protein. In this case, more SDS can be used (by using more or more concentrated
sample buffer) and the amount of protein in the sample application can be
reduced. An overloading of the gel with a soluble protein creates a semicircular
band of this protein (e. g. in the marker lane of the image at 66 KDa), allowing
other proteins with similar molecular weights to be covered. A low contrast (as
in the marker lane of the image) between bands within a lane indicates either the
presence of many proteins (low purity) or, if using purified proteins and a low
contrast occurs only below one band, it indicates a proteolytic degradation of the
protein, which first causes degradation bands, and after further degradation
produces a homogeneous color ("smear") below a band.[34] The documentation
of the banding pattern is usually done by photographing or scanning. For a
subsequent recovery of the molecules in individual bands, a gel extraction can be
performed.

Archiving
After protein staining and documentation of the banding pattern, the
Coomassie-stained 10% Tris/Tricine
polyacrylamide gel can be dried for archival storage. Proteins can be extracted gel. In the left lane, a molecular
from it at a later date. The gel is either placed in a drying frame (with or without weight size marker was used to
the use of heat) or in a vacuum dryer. The drying frame consists of two parts, estimate the size (from top to bottom:
one of which serves as a base for a wet cellophane film to which the gel and a 66, 45, 35, 24, 18 and 9 kDa). In the
remaining lanes purified yeast
one percent glycerol solution are added. Then a second wet cellophane film is
proteins were separated.
applied bubble-free, the second frame part is put on top and the frame is sealed
with clips. The removal of the air bubbles avoids a fragmentation of the gel
during drying. The water evaporates through the cellophane film. In contrast to
the drying frame, a vacuum dryer generates a vacuum and heats the gel to about
50 °C.

Molecular mass determination

For a more accurate determination of the molecular weight, the relative
migration distances of the individual protein bands are measured in the
separating gel.[35][36] The measurements are usually performed in triplicate for
Two SDS gels after completed
increased accuracy. The relative mobility (called Rf value or Rm value) is the
separation of the samples and
quotient of the distance of the band of the protein and the distance of the buffer staining in a drying frame
front. The distances of the bands and the buffer front are each measured from the
beginning of the separation gel. The distance of the buffer front roughly
corresponds to the distance of the bromophenol blue contained in the sample buffer. The relative distances of the proteins of the
size marker are plotted semi-logarithmically against their known molecular weights. By comparison with the linear part of the
generated graph or by a regression analysis, the molecular weight of an unknown protein can be determined by its relative
mobility. Bands of proteins with glycosylations can be blurred.[33] Proteins with many basic amino acids (e. g. histones)[37] can
lead to an overestimation of the molecular weight or even not migrate into the gel at all, because they move slower in the
electrophoresis due to the positive charges or even to the opposite direction.
Accordingly, many acidic amino acids can lead to accelerated migration of a
protein and an underestimation of its molecular mass.[38]

Applications
The SDS-PAGE in combination with a protein stain is widely used in
biochemistry for the quick and exact separation and subsequent analysis of
proteins. It has comparatively low instrument and reagent costs and is an easy-
to-use method. Because of its low scalability, it is mostly used for analytical
purposes and less for preparative purposes, especially when larger amounts of a
protein are to be isolated. The proteins of the size marker
(black X) show an approximately
Additionally, SDS-PAGE is used in combination with the western blot for the straight line in the representation of
determination of the presence of a specific protein in a mixture of proteins - or log M over Rf. The molecular weight
for the analysis of post-translational modifications. Post-translational of the unknown protein (red X) can
be determined on the y-axis.
modifications of proteins can lead to a different relative mobility (i.e. a band
shift) or to a change in the binding of a detection antibody used in the western
blot (i.e. a band disappears or appears).

In mass spectrometry of proteins, SDS-PAGE is a widely used method for sample preparation prior to spectrometry, mostly using
in-gel digestion. In regards to determining the molecular mass of a protein, the SDS-PAGE is a bit more exact than an analytical
ultracentrifugation, but less exact than a mass spectrometry or - ignoring post-translational modifications - a calculation of the
protein molecular mass from the DNA sequence.

In medical diagnostics, SDS-PAGE is used as part of the HIV test and to evaluate proteinuria. In the HIV test, HIV proteins are
separated by SDS-PAGE and subsequently detected by Western Blot with HIV-specific antibodies of the patient, if they are
present in his blood serum. SDS-PAGE for proteinuria evaluates the levels of various serum proteins in the urine, e.g. Albumin,
Alpha-2-macroglobulin and IgG.

Variants
SDS-PAGE is the most widely used method for gel electrophoretic separation of proteins. Two-dimensional gel electrophoresis
sequentially combines isoelectric focusing or BAC-PAGE with a SDS-PAGE. Native PAGE is used if native protein folding is to
be maintained. For separation of membrane proteins, BAC-PAGE or CTAB-PAGE may be used as an alternative to SDS-PAGE.
For electrophoretic separation of larger protein complexes, agarose gel electrophoresis can be used, e.g. the SDD-AGE. Some
enzymes can be detected via their enzyme activity by zymography.

Alternatives
While being one of the more precise and low-cost protein separation and analysis methods, the SDS-PAGE denatures proteins.
Where non-denaturing conditions are necessary, proteins are separated by a native PAGE or different chromatographic methods
with subsequent photometric quantification, for example affinity chromatography (or even tandem affinity purification), size
exclusion chromatography, ion exchange chromatography.[39] Proteins can also be separated by size in a tangential flow
filtration[40] or an ultrafiltration.[41] Single proteins can be isolated from a mixture by affinity chromatography or by a pull-down
assay. Some historically early and cost effective but crude separation methods usually based upon a series of extractions and
precipitations using kosmotropic molecules, for example the ammonium sulfate precipitation and the polyethyleneglycol
precipitation.
History
In 1948, Arne Tiselius was awarded the Nobel Prize in Chemistry for the discovery of the principle of electrophoresis as the
migration of charged and dissolved atoms or molecules in an electric field.[42] The use of a solid matrix (initially paper discs) in a
zone electrophoresis improved the separation. The discontinuous electrophoresis of 1964 by L. Ornstein and B. J. Davis made it
possible to improve the separation by the stacking effect.[43] The use of cross-linked polyacrylamide hydrogels, in contrast to the
previously used paper discs or starch gels, provided a higher stability of the gel and no microbial decomposition. The denaturing
effect of SDS in continuous polyacrylamide gels and the consequent improvement in resolution was first described in 1965 by
David F. Summers in the working group of James E. Darnell to separate poliovirus proteins.[44] The current variant of the SDS-
PAGE was described in 1970 by Ulrich K. Laemmli and initially used to characterise the proteins in the head of bacteriophage
T4.[1]

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External links
OpenWetWare: protocol for BisTris SDS-PAGE (http://openwetware.org/wiki/Sauer:bis-Tris_SDS-PAGE,_the_ver
y_best)

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