Download Lab Session 9

Lab Session 9
IUG, 2012
TMZ
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SDS-PAGE
Part I
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SDS-PAGE (PolyAcrylamide Gel
Electrophoresis)
• SDS-PAGE, sodium dodecyl sulfate
polyacrylamide gel
electrophoresis, is a technique
widely used in biochemistry,
forensics, genetics and molecular
biology:
• to separate proteins according to
their electrophoretic mobility (a
function of length of polypeptide
chain or molecular weight).
• to separate proteins according to
their size.
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Electrophoretic Theory
In an electric field:
• Charged molecules behave in a predictable
manner.
+ Positively charged molecules will move towards
the negative pole
+ Negatively charged molecules move towards
the positive pole.
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..SDS
• SDS (the detergent soap) breaks up hydrophobic areas
and coats proteins with negative charges thus
overwhelming positive charges in the protein.
• The detergent binds to hydrophobic regions in a
constant ratio of about 1.4 g of SDS per gram of
protein.
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Steps in SDS-PAGE
•
•
•
•
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Extract Protein
Solubilize and Denature Protein
Separate Proteins on a gel
Stain proteins (visualization)
Analyze and interpret results
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PAGE
• If the proteins are denatured and put into an
electric field (only), they will all move towards
the positive pole at the same rate, with no
separation by size.
• However, if the proteins are put into an
environment that will allow different sized
proteins to move at different rates.
• The environment is polyacrylamide.
• The entire process is called polyacrylamide
gel electrophoresis (PAGE).
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…In a polyacrylamide gel
• Small molecules move through the
polyacrylamide forest faster than big
molecules
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Why using Polyacrylamide?
• It is inert
• Can easily be made up at a different
concentrations to produce different pore sizes.
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…SDS-PAGE
• The end result of SDS-PAGE has two important
features:
 all proteins contain only primary structure
&
 all proteins have a large negative charge
which means they will all migrate towards the
positive pole when placed in an electric field.
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The actual bands are equal in size, but the
proteins within each band are of different sizes.
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Sample of SDS- PAGE
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Protein gel (SDS-PAGE) that has been
stained with Coomassie Blue.
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Polyacrylamide Gel
Cathode
Anode
Proteins separated by molecular weight
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The gel matrix
• Discontinuous
buffer system?
Laemmli buffer system:
Buffer in gel & tank are different
• A stacking gel at pH 6.8, buffered
by Tris-HCl
• A running gel buffered to pH 8.8
by Tris-HCl and an electrode buffer
at pH 8.3
Note: Stacking gel has a low conc. of
polyacrylamide, while the running gel has
a high conc., of polyacrylamide to slow
down the movement of proteins
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The role of Glycine..
• Glycine can exist in three different charge
states, positive, neutral or negative depending
on the pH.
• Control of the charge state of the glycine by
the different buffers is the key to the whole
stacking gel thing.
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The Way stacking gel works..
• When the power is turned on:
1. Negatively-charged glycine ions in the pH 8.3
electrode buffer are forced to enter the
stacking gel, where the pH is 6.8.

In this environment glycine switches
predominantly to the zwitterionic (neutrally
charged) state.
 This loss of charge causes them to move very
slowly in the electric field.
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2. The Cl- ions (from Tris-HCl) on the other hand,
move much more quickly in the electric field
and they form an ion front that migrates
ahead of the glycine.
3. The separation of Cl- from the Tris counter-ion
(which is now moving towards the cathode)
creates a narrow zone with a steep voltage
gradient that pulls the glycine along behind it,
resulting in two narrowly separated fronts of
migrating ions;
 the highly mobile Cl- front, followed by the
slower, mostly neutral glycine front.
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4. All of the proteins in the gel sample have an
electrophoretic mobility that is intermediate
between the extreme of the mobility of the
glycine and Clso when the two fronts sweep through the sample
well the proteins are concentrated into the narrow
zone between the Cl- and glycine fronts.
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5. This procession carries on until it hits the running
gel,
where the pH switches to 8.8. At this pH the glycine
molecules are mostly negatively charged and can
migrate much faster than the proteins.
So the glycine front accelerates past the proteins,
leaving them behind.
6. The result is that the proteins are dumped in a
very narrow band at the interface of the stacking
and running gels.
 Since the running gel has an increased acrylamide
concentration, which slows the movement of the
proteins according to their size, the separation
begins…
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What if not using a stacking gel?
• In the absence of a stacking gel, your sample
would sit on top of the running gel, as a band of
up to 1cm deep.
• Rather than being lined up together and hitting
the running gel together, the proteins in the
sample would all enter the running gel at
different times, resulting in very smeared bands.
• So the stacking gel ensures that all of the proteins
arrive at the running gel at the same time so
proteins of the same molecular weight will
migrate as tight bands.
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The Running Gel..
• Separation
• Once the proteins are in the running gel, they are
separated because higher molecular weight
proteins move more slowly through the porous
acrylamide gel than lower molecular weight
proteins.
• The size of the pores in the gel can be altered
depending on the size of the proteins you want to
separate by changing the acrylamide
concentration.
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Kaleidoscope Standard
MW (daltons)
on Tris HCl
gel
Protein
Color
Myosin
Blue
204,649
B-galactosidase
Magenta
127,511
Bovine serum
albumin
Green
85,130
Carbonic
anhydrase
Violet
37,830
Soybean
trypsin
inhibitor
Orange
30,906
Lysozyme
Red
27,230
Aprotinin
Blue
6,638
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Polyacrylamide gel polymerisation
• Polyacrylamide, used mainly for SDS-PAGE,
• Is a matrix formed from monomers of
acrylamide and bis-acrylamide.
• It’s strengths are that is it chemically inert – so
won’t interact with proteins as they pass
through – and that it can easily and
reproducibly be made with different pore sizes
to produce gels with different separation
properties.
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• The polymerisation reaction, shown in the
diagram below, is a vinyl addition catalysed by
free radicals.
• The reaction is initiated by TEMED, which
induces free radical formation from
ammonium persulphate (APS).
• The free radicals transfer electrons to the
acrylamide/bisacrylamide monomers,
radicalizing them and causing them to react
with each other to form the polyacrylamide
chain.
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• In the absence of bis-acrylamide, the acrylamide
would polymerise into long strands, not a porous
gel.
• But as the diagram shows, bis-acrylamide crosslinks the acrylamide chains and this is what gives
rise to the formation of the porous gel matrix.
• The amount of crosslinking, and therefore the
pore size and consequent separation properties
of the gel can be controlled by varying the ratio of
acrylamide to bis-acrylamide.
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