Transcript SDS Polyacrylamide Gel Electrophoresis

Polyacrylamide Gel
Electrophoresis
Dr. Nikhat Siddiqi
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• The most widely-used polysaccharide gel matrix nowadays
is that formed with agarose. This is a polymer composed of
a repeating disaccharide unit called agarobiose which
consists of galactose and 3, 6-anhydrogalactose.
• Agarose gives a more uniform degree of porosity than
starch and this may be varied by altering the starting
concentration of the suspension (low concentrations give
large pores while high concentrations give smaller pores).
• This gel has found widespread use especially in the
separation of DNA molecules (although it may also be used
in some electrophoretic procedures involving protein
samples such as immunoelectrophoresis.
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• Polyacrylamide gels, formed by the
polymerization of acrylamide and cross-linked
by methylenebisacrylamide, are choice
supporting media for electrophoresis because
they are chemically inert and are readily
formed.
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• A far stronger gel suitable for electrophoretic separation of both
proteins and nucleic acids may be formed by the polymerization of
acrylamide.
• The inclusion of a small amount of acrylamide crosslinked by a
methylene bridge (N,N methylene bisacrylamide) allows formation
of a crosslinked gel with a highly-controlled porosity which is also
mechanically strong and chemically inert.
• For separation of proteins, the ratio of acrylamide: N,N methylene
bisacrylamide is usually 40 : 1 while for DNA separation it is 19 : 1.
• Such gels are suitable for high resolution separation of DNA and
proteins across a large mass range .
• A wider range of molecular mass in an individual gel is achievable
by the use of gradient gels. These consist of a gradient of
polyacrylamide (e.g. 5–20%).
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Polymerisation of
acrylamide to form polyacrylamide gel.
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Polyacrylamide Nondenaturing
Electrophoresis
• The gel network formed by polyacrylamide is a
suitable environment for the electrophoretic
separation of proteins in their native state,
that is under nondenaturing conditions.
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DENATURING ELECTROPHORESIS
• Such experiments are especially useful in the
study of proteins since they have a more
varied range of tertiary structure than nucleic
acids.
• Any biological activity or quaternary structure
associated with the sample components is lost
in denaturing electrophoresis. However,
important structural information can be
obtained.
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SDS Polyacrylamide Gel
Electrophoresis
• The detergent sodium dodecyl sulphate (SDS) consists of a
hydrophobic 12-carbon chain and a polar sulfated head.
• The hydrophobic chain can intercalate into hydrophobic
parts of the protein by detergent action, disrupting its
compact folded structure.
• SDS coats proteins with a uniform ‘layer’ of negative
charges which causes them to migrate towards the anode
when placed in an electrical field, regardless of the net
intrinsic charge of the uncomplexed proteins.
• The negative charge gives a charge density largely
independent of the primary structure or mass of the
polypeptide.
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• Proteins can be separated largely on the basis
of mass by electrophoresis in a polyacrylamide
gel under denaturing conditions.
• Mercaptoethanol (2-thioethanol) or
dithiothreitol also is added to reduce disulfide
bonds.
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• The SDS-protein complexes are then subjected to
electrophoresis.
• When the electrophoresis is complete, the
proteins in the gel can be visualized by staining
them with silver or a dye such as Coomassie blue,
which reveals a series of bands.
• Radioactive labels can be detected by placing a
sheet of x-ray film over the gel, a procedure
called autoradiography.
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Staining of Proteins After
Electrophoresis
• Proteins subjected to electrophoresis on an
SDS-polyacrylamide gel can be visualized by
staining with Coomassie blue.
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• Although most proteins stain with this dye, it should be
noted that not all proteins take up the dye with equal
affinity.
• Less sensitive protein dyes include ponceau red and amido
black. Ponceau red has the advantage that it stains
reversibly and may be removed from the protein to allow
subsequent analysis.
• The most sensitive staining method for protein is silver
staining. This involves soaking the gel in AgNO3 which
results in precipitation of metallic silver (Ag0) at the
location of protein.
• Radioactive proteins/nucleic acids can be visualized after
electrophoretic separation by autoradiography.
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Electrophoresis Can Determine Mass
• The electrophoretic
mobility of many
proteins in SDSpolyacrylamide gels is
inversely proportional
to the logarithm of their
mass. (K. Weber and M.
Osborn).
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Discontinuous polyacrylamide gel
electrophoresis
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• Stacking gel has a low concentration of polyacrylamide (3–
5%), low ionic strength and a pH near neutrality.
• The resolving gel, by contrast, is composed of a higher
percentage of acrylamide (8–20%), higher ionic strength
and an alkaline pH.
• This gel achieves separation of sample molecules stacked at
the interface. The lower ionic strength of the stacking gel
causes higher electrical resistance and hence stronger
electrical field strength in this gel compared to the
resolving gel.
• This means that, at a given voltage, samples have higher
mobility in the stacking compared to the resolving gel.
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Isoelectric Focussing
• Suppose that a mixture of proteins undergoes
electrophoresis in a pH gradient in a gel in the absence of
SDS.
• Each protein will move until it reaches a position in the gel
at which the pH is equal to the pI of the protein.
• This method of separating proteins according to their
isoelectric point is called isoelectric focusing.
• The pH gradient in the gel is formed first by subjecting a
mixture of polyampholytes (small multicharged polymers)
having many pI values to electrophoresis.
• Isoelectric focusing can readily resolve proteins that differ
in pI by as little as 0.01, which means that proteins differing
by one net charge can be separated.
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Isoelectric Focusing
• A pH gradient is
established in a gel before
loading the sample.
• (A) The sample is loaded
and voltage is applied.
The proteins will migrate
to their isoelectric pH, the
location at which they
have no net charge.
• (B) The proteins form
bands that can be excised
and used for further
experimentation.
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