Chromatography High Performance Liquid Chromatography HPLC Chapter 4-5 1 Dr Gihan Gawish.
Dr Gihan Gawish 1 PAGE. Types of Gels Dr Gihan Gawish The most common types of gels are: Starch...
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Transcript of Dr Gihan Gawish 1 PAGE. Types of Gels Dr Gihan Gawish The most common types of gels are: Starch...
Dr Gihan Gawish
1
PAGE
Types of Gels
Dr Gihan Gawish
The most common types of gels are: Starch gels: seldom used nowadays
Agarose gels: for separation of nucleic acids and large proteins
Polyacrylamide gels: for separation of most proteins and small nucleic acids
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Dr Gihan Gawish
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Polyacrylamide Gel Electrophoresis (PAGE)
PAGE can be classified according the separation conditions into:
1- Native-PAGE: Separation is based upon charge, size, and shape of
macromolecules. Useful for separation and/or purification of mixture of
proteins This was the original mode of electrophoresis
(introduced in 1930s, Nobel Prize 1948).
2- Denatured-PAGE or SDS-PAGE Separation is based upon the molecular weight of
proteins. The most common method for determining MW of
proteins Very useful for checking purity of protein samples
PAGE
Dr Gihan Gawish
3- Isoelectric Focusing-PAGE Separation of basis of pI, not MW Recently, became popular as a part of proteomic
techniques
PAGE can also be classified according to the physical shape of the gel:
slab (most common) or tubes Continuous, discontinuous, stacked, or gradient gel One dimensional or two-dimensional
electrophoresis
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Dr Gihan Gawish
Biopolymers such as proteins and nucleic acids are
folded into compact structures
They held together by a variety of non- covalent,
ionic interactions such as hydrogen bonding
salt bridges.
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SDS-PAGE
The electrophoretic mobility of the denaturated
molecule will be changed, compared to that in
non denaturating conditions
It migrates as an unstructured monomer through
the electrical field.
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SDS-PAGE
SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
Dr Gihan Gawish
SDS-PAGE, is a technique widely used to
separate proteins according to their
electrophoretic mobility
The SDS gel electrophoresis of samples
having: identical charge to mass ratios
results in fractionation by size
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SDS-PAGE
Dr Gihan Gawish
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Dr Gihan Gawish
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SDS-PAGE
Dr Gihan Gawish
All proteins are made to look virtually the same rod diameter but with lengths that are
proportional to the molecular weight of the protein.
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SDS-PAGE: Procedure
Dr Gihan Gawish
The solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures
secondary and non–disulfide–linked tertiary structures, and applies a 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
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.
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SDS PAGE in practice
Dr Gihan Gawish
Denatured SDS-protein mixture with a colored dye or stain added for tracking is loaded at the top of a slab or tube of a gel (typically
polyacrylamide)
Electric field imposed within the gel using electrodes attached to a power supply
Proteins and dye migrate down the gel at a constant rate that depends on the molecular
weight of the protein Smaller proteins migrating faster
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SDS PAGE in practice
Dr Gihan Gawish
Over a limited molecular
weight range, the
electrophoretic mobility of
proteins is found to be
proportional to the logarithm
of their molecular weight
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SDS- PAGE Advantages
Dr Gihan Gawish
Rapidly and cheaply measure molecular weights with an accuracy of about 5%
determine trace amounts of impurities in a sample
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Preparative SDS-PAGE
Dr Gihan Gawish
Samples are electrophoresed (native or SDS PAGE) through a
cylindrical gel
Bands pass through a thin frit within the
elution chamber
Isolated bands are drawn radially by a
pump onto a fraction collector into discreet
liquid fractions
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Isoelectric focusing-PAGE
Dr Gihan Gawish
Isoelectric focusing is a technique for separating different molecules by their
electric charge differences.
The charge of molecule changes with the pH of its surroundings.
A protein that is in a pH region below its isoelectric point (pI) will be positively
charged and so will migrate towards the negative electrolode.
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Employs a pH gradient extending the entire gel: (slabs or tubes)
Carrier ampholytes are
used to set up the pH gradient
Protein sample is applied:
no SDS. charges make proteins different
Isoelectric Focusing (IEF)-PAGE
IEF-PAGE At pH = pI, a protein will have no net charge
stop moving At any other pH in the gradient, the protein has either a
positive charge (pH<pI) or negative charge (pH>pI) Runs requires higher voltages and longer periods
of time, but gives resolution up ±0.001 pH
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