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Gel electrophoresis of proteins

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Proteins separated by SDS-PAGE, Coomassie Brilliant Blue staining


Protein electrophoresis is a method for analysing the proteins in a fluid or an extract. The
electrophoresis may be performed with a small volume of sample in a number of alternative ways with
or without a supporting medium: SDS polyacrylamide gel electrophoresis (in short: gel electrophoresis,
PAGE, or SDS-electrophoresis), free-flow electrophoresis, electrofocusing, isotachophoresis, affinity
electrophoresis, immunoelectrophoresis, counterelectrophoresis, and capillary electrophoresis. Each
method has many variations with individual advantages and limitations. Gel electrophoresis is often
performed in combination with electroblotting immunoblotting to give additional information about a
specific protein. Because of practical limitations, protein electrophoresis is generally not suited as a
preparative method.

Contents [hide]
1

Denaturing gel methods

1.1

SDS-PAGE

Native gel methods

2.1

Blue native PAGE

2.2

Clear native PAGE

2.3

Quantitative preparative native continuous PAGE

Buffer systems

3.1

SDS gradient gel electrophoresis of proteins

Visualization

Medical applications

See also

References

External links

Denaturing gel methods[edit]


SDS-PAGE[edit]
Main article: SDS-PAGE
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, describes a collection of related
techniques to separate proteins according to their electrophoretic mobility (a function of the length of a
polypeptide chain and its charge) while in the denatured (unfolded) state. 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.

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. In this process, the intrinsic charges of polypeptides becomes negligible when

compared to the negative charges contributed by SDS. Thus polypeptides after treatment become rodlike structures possessing a uniform charge density, that is same net negative charge per unit length.
The electrophoretic mobilities of these proteins will be a linear function of the logarithms of their
molecular weights.

Native gel methods[edit]


Native gels, also known as non-denaturing gels, analyze proteins that are still in their folded state.
Thus, the electrophoretic mobility depends not only on the charge-to-mass ratio, but also to the
physical shape and size of the protein.

Blue native PAGE[edit]


BN-PAGE is a native PAGE technique, where the Coomassie Brilliant Blue dye provides the necessary
charges to the protein complexes for the electrophoretic separation.[1][2] The disadvantage of
Coomassie is that in binding to proteins it can act like a detergent causing complexes to dissociate.
Another drawback is the potential quenching of chemoluminescence (e.g. in subsequent western blot
detection or activity assays) or fluorescence of proteins with prosthetic groups (e.g. heme or
chlorophyll) or labelled with fluorescent dyes.

Clear native PAGE[edit]


CN-PAGE (commonly referred to as Native PAGE) separates acidic water-soluble and membrane proteins
in a polyacrylamide gradient gel. It uses no charged dye so the electrophoretic mobility of proteins in
CN-PAGE (in contrast to the charge shift technique BN-PAGE) is related to the intrinsic charge of the
proteins.[3] The migration distance depends on the protein charge, its size and the pore size of the gel.
In many cases this method has lower resolution than BN-PAGE, but CN-PAGE offers advantages
whenever Coomassie dye would interfere with further analytical techniques, for example it has been
described as a very efficient microscale separation technique for FRET analyses.[4] Also CN-PAGE is
milder than BN-PAGE so it can retain labile supramolecular assemblies of membrane protein complexes
that are dissociated under the conditions of BN-PAGE.

Quantitative preparative native continuous PAGE[edit]


Main article: QPNC-PAGE
In contrast to CN-PAGE and BN-PAGE the folded protein complexes of interest separate cleanly and
predictably, since they move through the polyacrylamide gel as quickly as individual, denatured
proteins under non-restrictive conditions. The separated proteins are continuously eluted into a
physiological eluent and transported to a fraction collector. In a few specific PAGE fractions metal
cofactors can be identified and quantified by high-resolution techniques like ICP-MS. The natural
structures of the isolated metalloproteins are elucidated by solution NMR.[5]

Buffer systems[edit]

Postulated migration of proteins in a Laemmli gel system A: Stacking gel, B: Resolving gel, o: sample
application c: discontinuities in the buffer and electrophoretic matrix
Most protein separations are performed using a "discontinuous" (or DISC) buffer system that
significantly enhances the sharpness of the bands within the gel. During electrophoresis in a

discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all
of the proteins to focus into a single sharp band. The formation of the ion gradient is achieved by
choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDScoated proteins. These conditions provide an environment in which Kohlrausch's reactions determine
the molar conductivity. As a result, SDS-coated proteins are concentrated to several fold in a thin zone
of the order of 19 m within a few minutes. At this stage all proteins migrate at the same migration
speed by isotachophoresis. This occurs in a region of the gel that has larger pores so that the gel matrix
does not retard the migration during the focusing or "stacking" event.[6][7] Separation of the proteins
by size is achieved in the lower, "resolving" region of the gel. The resolving gel typically has a much
smaller pore size, which leads to a sieving effect that now determines the electrophoretic mobility of
the proteins. At the same time, the separating part of the gel also has a pH value in which the buffer
ions on average carry a greater charge, causing them to "outrun" the SDS-covered proteins and
eliminate the ion gradient and thereby the stacking effect.

A very widespread discontinuous buffer system is the tris-glycine or "Laemmli" system that stacks at a
pH of 6.8 and resolves at a pH of ~8.3-9.0. A drawback of this system is that these pH values may
promote disulfide bond formation between cysteine residues in the proteins because the pKa of
cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate
with the proteins. Recent advances in buffering technology alleviate this problem by resolving the
proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g.
sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An
additional benefit of using buffers with lower pH values is that the acrylamide gel is more stable at
lower pH values, so the gels can be stored for long periods of time before use.[8][9]

SDS gradient gel electrophoresis of proteins[edit]


As voltage is applied, the anions (and negatively charged sample molecules) migrate toward the
positive electrode (anode) in the lower chamber, the leading ion is Cl ( high mobility and high
concentration); glycinate is the trailing ion (low mobility and low concentration). SDS-protein particles
do not migrate freely at the border between the Cl of the gel buffer and the Gly of the cathode
buffer. Friedrich Kohlrausch found that Ohm's law also applies to dissolved electrolytes. Because of the
voltage drop between the Cl and Glycine-buffers, proteins are compressed (stacked) into micrometer
thin layers.[10] The boundary moves through a pore gradient and the protein stack gradually disperses
due to a frictional resistance increase of the gel matrix. Stacking and unstacking occurs continuously in
the gradient gel, for every protein at a different position. For a complete protein unstacking the
polyacrylamide-gel concentration must exceed 16% T. The two-gel system of "Laemmli" is a simple
gradient gel. The pH discontinuity of the buffers is of no significance for the separation quality, and a
"stacking-gel" with a different pH is not needed.

Visualization[edit]
The most popular protein stain is Coomassie Brilliant Blue. 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.

When more sensitive method than staining by Coomassie is needed silver staining is usually used.
Silver staining is a sensitive procedure to detect trace amounts of proteins in gels, but can also
visualize nucleic acid or polysaccharides.

Similarly as in nucleic acid gel electrophoresis, tracking dye is often used. Anionic dyes of a known
electrophoretic mobility are usually included in the sample buffer. A very common tracking dye is
Bromophenol blue. 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.

Medical applications[edit]

Schematic representation of a protein electrophoresis gel.

Serum protein electrophoresis showing a paraprotein (peak in the gamma zone) in a patient with
multiple myeloma.
Main articles: Serum protein electrophoresis and Blood proteins
In medicine, protein electrophoresis is a method of analysing the proteins mainly in blood serum.
Before the widespread use of gel electrophoresis, protein electrophoresis was performed as free-flow
electrophoresis (on paper) or as immunoelectrophoresis.

Traditionally, two classes of blood proteins are considered: serum albumin and globulin. They are
generally equal in proportion, but albumin as a molecule is much smaller and lightly, negativelycharged, leading to an accumulation of albumin on the electrophoretic gel. A small band before albumin
represents transthyretin (also named prealbumin). Some forms of medication or body chemicals can
cause their own band, but it usually is small. Abnormal bands (spikes) are seen in monoclonal
gammopathy of undetermined significance and multiple myeloma, and are useful in the diagnosis of
these conditions.

The globulins are classified by their banding pattern (with their main representatives):

The alpha () band consists of two parts, 1 and 2:


1 - 1-antitrypsin, 1-acid glycoprotein.
2 - haptoglobin, 2-macroglobulin, 2-antiplasmin, ceruloplasmin.
The beta () band - transferrin, LDL, complement
The gamma () band - immunoglobulin (IgA, IgD, IgE, IgG and IgM). Paraproteins (in multiple myeloma)
usually appear in this band.
Normal present medical procedure involves determination of numerous proteins in plasma including
hormones and enzymes, some of them also determined by electrophoresis. However, gel
electrophoresis is mainly a research tool, also when the subject is blood proteins.