Chemistry 4010 Lab
 It’s
all about
PROTEINS…
SDS-PAGE

Understanding SDS and PAGE
– SDS- Sodium Dodecyl Sulfate
a detergent soap that dissolves hydrophobic
molecules
 also has a negative charge
 by incubating the cell with SDS, the
membranes are dissolved and proteins are
solubilized
 protein now possesses negative charges

– PAGE- Polyacrylamide Gel Electrophoresis
SDS-PAGE

Understanding SDS and PAGE
– SDS- Sodium Dodecyl Sulfate
– PAGE- Polyacrylamide Gel Electrophoresis
polymer of acrylamide monomers
 with a mixture of other buffers and solutions
this polymer can be changed into a gel matrix
 electricity is used to pull the protein through
the gel
 the negatively charged protein moves to the
positive poles

Side view
Top View
How does it all work

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add the proteins to the gel matrix
turn on the current
negatively charged proteins will move
through the gel
smaller sized proteins will move at a
faster rate due to their ability to
maneuver through the gel
Proteins Moving through
the Gel
What will the gel look like


smaller proteins will
move through the
gel faster while
larger proteins
move at a slower
pace
Visualize the bands
by staining the gel
with Coomassie
Blue
SDS- PAGE Setup
SDS-PAGE vs. Gel
Electrophoresis

SDS-PAGE is a
great tool for
analyzing proteins,
whereas Gel
Electrophoresis is
used to study DNA.
Gel Electrophoresis

Makes use of an agarose gel
– Agarose is a linear polysaccharide
(average molecular mass about 12,000)
made up of the basic repeat unit
agarobiose, which comprises alternating
units of galactose and 3,6anhydrogalactose. Agarose is usually
used at concentrations between 1% and
3%.
Gel Electrophoresis

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DNA is cut by restriction enzymes to
yield many different sized pieces of
DNA
Like SDS-PAGE, an electric current is
applied and the negatively charged
DNA moves through the gel
Smaller pieces move faster
Gel Electrophoresis Setup
THEORY
SDS-PAGE & Gel Electrophoresis

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These techniques make use of the fact that molecules are being
separated based on size and charge, based on the following equations
v = the rate (velocity) of migration
E = the strength of the electrical field
z = the charge on the molecule
f = the frictional force on the molecule
Frictional force can then be defined as
η is the viscosity of the medium
r is the stokes radius of the molecule
Gel Filtration or GPC
 Gel filtration
chromatography is a
separation based on size
–stationary phase consists of
porous beads with a welldefined range of pore sizes
–mobile phase consists of the
solvent
GPC

Proteins that are too large to fit inside any of the
pores-EXCLUDED
– access only to the mobile phase between the beads and
elute first.

Proteins of intermediate size –PARTIALLY
INCLUDED
– fit inside some but not all of the pores in the beads. These
proteins will then elute between the large ("excluded")
and small ("totally included") proteins

Proteins that are small enough can fit inside all the
pores in the beads -INCLUDED
– access to the mobile phase inside the beads as well as the
mobile phase between beads and elute last
Theory of GPC

These separations can be described by this
equation
– Vr = the retention volume of the protein
– Vo = the volume of mobile phase between the
beads of the stationary phase inside the column
(sometimes called the void volume)
– Vi = the volume of mobile phase inside the
porous beads (also called the included volume)
– K = the partition coefficient (the extent to which
the protein can penetrate the pores in the
stationary phase, with values ranging between 0
and 1).
GPC Setup
Conclusion
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All of these techniques serve to
enhance our ability to better elucidate
the size, charge and polarity of
polymers in science
Understanding the parallel nature of
these experiments will allow us to
bridge the gap between natural and
synthetic polymers