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Why study proteins?
• Proteins are fascinating molecular
devices. They play important roles in the
life processes. There are structural
proteins , catalytic proteins, transport and
storage proteins, regulatory proteins,
proteins of the immune system and the
immunoglobulin superfamily, cell-cell
recognition and signaling proteins.
Estimation of Proteins
Assay of Protein Purity, Molecular
Weight, and Subunit Structure
SDS Polyacrylamide Gel
Electrophoresis of Proteins
• SDS-PAGE is the most widely used
method for qualitatively analyzing protein
mixtures. It is particularly useful for
monitoring protein purification, and
because the method is based on the
separation of proteins according to size,
the method can also be used to determine
the relative molecular mass of proteins.
Formation of Polyacrylamide Gels:
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Crosslinked polyacrylamide gels are formed from the
polymerization of acrylamide monomer in the presence
of N,N'-methylene-bis-acrylamide .
Bisacrylamide is essentially two acrylamide molecules
linked by a methylene group and is used as a
crosslinking agent.
Acrylamide monomer is polymerized in a head-to-tail
fashion into long chains, and occasionally a bisacrylamide molecule is built into the growing chain,
thus introducing a second site for chain extension.
Proceeding in this way, a crosslinked matrix of fairly
well-defined structure is formed.
Polymerization of Acrylamide
The polymerization of acrylamide is an example of free-radical catalysis,
and is initiated by the addition of ammonium persulfate and the base
N,N,N',N'-tetramethylenediamine (TEMED). TEMED catalyzes the
decomposition of the persulfate ion to give a free radical (a molecule with
an unpaired electrons.
The Use of Stacking Gels
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The purpose of the stacking gel is to concentrate the protein sample into a sharp band before it
enters the mail separating gel, thus giving sharper protein bands in the separating gel.
This modification allows relatively larger sample volumes to be applied to the gel without any loss
of resolution.
The stacking gel has a very large pore size (4% acrylamide) which allows the proteins to move
freely and concentrate, or stack under the effect of the electric field.
Sample concentration is produced by isotachophoresis (the band-sharpening effect) of the sample
in the stacking gel.
The isotachophoresis relies on the fact that the negatively charged glycinate ions (in the reservoir
buffer) have a lower electrophoretic mobility than the protein-SDS complexes, which in turn have
lower mobility than the chloride ions if they are in a region of higher field strength.
Field strength is inversely proportional to conductivity, which is proportional to concentration. The
result is that the three species of interest adjust their concentrations so that [chloride ion]>[proteinSDS]>[glycinate].
There are only a small quantity of protein-SDS complexes, so they concentrate in a very tight
band between the glycinate and chloride ions boundaries. Once the glycinate reaches the
separating gel, it becomes more fully ionized in the higher pH environment and its mobility
increases. (the pH of the stacking gel is 6.8 and that of the separating gel is 8.8).
Thus the interface between glycinate and chloride ions leaves behind the protein-SDS complexes,
which are left to electrophorese at their own rates.
SDS-PAGE
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Samples to be run on SDS-PAGE are first boiled in sample buffer containing βmercaptoethanol or DTT (Dithiothreitol) and SDS. The mercaptoethanol reduces any
disulfide bridges present that are holding together the protein tertiary structure. SDS
is an anionic detergent and binds strongly to, and denatures , the proteins.
Each protein in the mixture is therefore fully denatured by this treatment and opens
up into a rod-shaped structure with a series of negatively charged SDS molecules
along the polypeptide chain.
On average, one SDS molecule binds for every two amino acid residues.
The sample buffer also contains an ionizable tracking dye usually bromophenol blue
that allows the elctrophoretic run to be monitored. Sucrose or glycerol which gives
the sample solution density. Thus allowing the sample to settle easily through the
electrophoresis buffer.
Silver-Stained SDS-PAGE
Standard Curve for the Estimation
of Protein MWs by SDS-PAGE
Gradient SDS-PAGE
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The use of PAGE that have a gradient of increasing acrylamide concentration (and
hence decreasing pore size) can sometimes have advantages over fixedconcentration acrylamide gels.
There are two main advantages of gradient gels over linear gels.
– A much greater range of protein Mr values can be separated than on a fixed
percentage gels. In a complex mixture, very low-mol-wt proteins travel freely
through the gel to begin with, and start to resolve when they reach the smaller
pore size toward the lower part of the gel. Much larger proteins can still enter the
gel but start to separate immediately owing to the sieving effect of the gel.
– Proteins with very similar Mr values may be resolved that can not be resolved in
fixed percentage gels. As each protein moves through the gel, the pore size
become smaller until the protein reaches its pore size limit. The pore size in the
gel is now too small to allow passage of the protein, and the protein sample
stacks up at this point as a sharp band. A similar-sized protein, but with slightly
lower Mr, will be able to travel a little further through the gel before reaching its
pore size limit, at which point it will form a sharp band. These two proteins, of
slightly different Mr values, therefore separate as two, close, sharp bands.
Silver Stained SDS-PAGE
Gradient forming Apparatus
Gelatin-SDS-PAGE Zymography
Zymography is a method for the detection of a specific enzyme among the bands separated by
electrophoresis.
Isoelectric Focusing (IEF)
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A protein has charged groups of both
polarities and therefore has an isoelectric
point, pI, at which it is immobile in an electric
field. If a mixture of proteins is
electrophoresed through a solution or gel
that has a stable pH gradient in which the
pH smoothly increases from anode to
cathode, each protein will migrate to the
position in the pH gradient corresponding to
its pI.
If proteins are distributed throughout a
solution in a pH gradient, then upon
application of an electric potential across the
gradient, with the anode at the low pH end,
molecules in the low pH zone (which will be
positively charged) will migrate to the
cathode. Conversely, molecules in the high
pH zone will be negatively charged and will
consequently migrate, through zones of
decreasing pH, towards the anode.
When each protein reaches a position where
the pH is equal to its pI, it will lose all of its
charge and its migration will cease.
2-D Electrophoresis
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In 2-D electrophoresis, proteins
are separated in the first
dimension, according to their
isoelectric point by IEF, and then
the separated proteins are
subjected to SDS-PAGE in the
perpendicular direction. Twodimensional PAGE is a valuable
tool for proteomics, a field of study
that involves cataloguing all of a
cell’s exposed proteins with
emphasis on their characterization
and functional activities.
Western Blots
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Immunoblotting (Alternatively,
Western blotting) is a method to
detect a specific protein in a given
sample of tissue homogenate or
extract. It uses SDS-PAGE to
separate polypeptides. Then, the
polypeptides are
electrophoretically transferred to a
membrane ( typically nitrocellulose
or PVDF). The transferred
proteins are bound to the surface
of the membrane where they are
probed using antibodies specific to
the target protein.
Immunoblots
Protein-Protein Interaction
The interactions between proteins are important for many biological
functions.
Signals from the exterior of a cell are mediated to the inside of that cell by protein-protein
interactions of the signalling molecules. This process, called signal transduction,
plays an important role in many biological processes and in many diseases.
Proteins might interact for a ling time to form part of a protein complex, a protein may be
carrying another protein ( for example, from cytoplasm to nucleus or vice versa in the
case of the nuclear pore importins).
A protein may interact briefly with another protein for the modification ( for example, a
protein kinase will add a phosphate to a target protein. This modification of proteins
can itself change protein-protein interaction. For example, some proteins with SH2
domains only bind to other proteins when they are phosphorylated on the amino acid
tyrosine.
In conclusion, protein-protein interactions are of central importance for virtually every
process in a living cell. Information about these interactions improves our
understanding of diseases and can provide the basis for new therapeutic approaches.
Methods to investigate protein-protein interactions
• Co-immunoprecipitation is considered to be good standard assay for
protein-protein interactions, especially when it is performed with
endogeneous (not over expressed and not tagged) proteins. The
protein of interest is isolated with a specific antibody. Interaction
proteins which adhere to this protein are subsequently identified by
Western blotting. Interactions detected by this method are
considered to be real. However, this method can only verify
interactions between suspected interaction partners. Thus, it is not
a screening approach.
Identification of Ras-associated proteins by coimmunoprecipitation
analysis
Coimmunoprecipitation Analysis of Cauda Epididymal
Fluid
Continued..
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Fluorescence resonance energy transfer (FRET) is a common technique when
observing the interactions of only two different proteins.
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Pull down assays are a common variation of IP and are used identically. A pull-down
assay is distinct from IP in that it requires ligands other than an antibody to capture
the protein complex.
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The yeast two-hybrid screen reveals the interaction between artificial fusion proteins
inside the nucleus of yeast. This approach can identify partners of a protein in an
unbiased manner. However, the method has a high false-positive rate which makes it
necessary to verify the identified interactions by co-IP.
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Chemical cross linking followed by High mass MALDI mass spectrometry can be
used to analyze intact protein interactions in place before trying to isolate/identify
interacting proteins. This method detects interactions among non-tagged proteins.