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0
=
e
Where c is the dielectric constant of the
solvent, c
0
is the permitivity of the vaccum
(8.85*10
-12
) and q is the dynamic vicocity
So far we have assumed that we are dealing with particles
whose size is on the order of the double layer. But our
analytes are much bigger. In this case, we are more
concerned with the stokes radius relative to the amount of
charge.
r
z
u
e
tq 6
=
Where z is the charge, q is the
viscosity and r is the Stokes radius
Electrophoresis of Nucleic Acids
If were trying to separate nucleic acids, we have a problem
because:
They are always negatively charged at neutral pH.
Each additional nucleoside confers an additional charge,
so charge is directly proportional to size.
All molecules will travel in the same direction
All molecules will have the same
e
The solution is to use a gel which consists of pores surrounded
by cross-linked fibers
EM image of
Agarose Gel
This will make
e
dependent
on the Stokes radius
DNA/RNA Electrophoresis
Double stranded DNA or RNA are molecules that repel
themselves. They will all form rod-like structures.
Single stranded DNA or RNA may interact with itself
forming a supercoil.
e
for supercoiled DNA or RNA is a
mite unpredictable.
So now we can separate our nucleic acids on the basis of
size. We can visualize with a fluorescent dye (usually
ethidium bromide) and compare to a standard to get a
relatively good quantitative size:
Electropherogram
Protein Electrophoresis
Proteins are even trickier than DNA/RNA:
They are all effectively supercoiled (3 Structure)
They can be either positively or negatively charged
The number of charges depends on the amino acid sequence
and is not proportional to size.
-ve
+ve
The solution is to expose
the protein so a detergent
(usually sodium dodecyl
sulphate - SDS)
Protein Electrophoresis
Proteins will bind an amount of SDS roughly proportional to
their size (1.4g/g polypeptide), so we have effectively
created the same situation as for DNA/RNA electrophoresis.
The gel of choice for SDS-page
protein electrophoresis is
polyacrylamide. Danger!
Acrylamide monomers are
potent neurotoxins
The
Standard
ladder
The Over-
Expressed
protein of
interest?
Proteins are visualized using a dye (coomassie blue)
or other stain (silver)
Sometimes proteins are pre-
boiled in a reducing agent to
eliminate S-S.
2D Electrophoresis
If you want to analyze the whole protein content of a cell, a
1D separation just isnt gonna do it.
The solution is to do a 2D separation using a gel
that has a pH gradient.
Proteins will run on this gel (in both directions)
until they hit their isoelectric point where they
aggregate
-ve
+ve
Then you can soak the gel in
SDS, flip the voltage 90 and
separate by size.
2D Gels
2D Electrophoresis is a powerful tool for analyzing the
protein complement of simple cells
Capillary Electrophoresis
You can also make molecules pass through a narrow (usually
fused silica) capillary
Typically, your analyte is in a buffer with net +ve charge,
which generates an electroosmotic flow (EOF) towards the
cathode
EOF overpowers
e
, thus all analytes move towards the
cathode at rates dependent on their
e
Capillary Electrophoresis
The advantages of CE are:
Really good separation of slow diffusing analytes
Very, very low sample consumption
|
|
|
|
|
.
|
\
|
+
A
=
) ( 4
1
o e
e
e
sep
D
V
R
Where: A
e
is the difference in
apparent electrophoretic mobility,
is the average electrophoretic
mobility, V is the applied voltage
and D is the diffusion coefficient
Resolution is limited by Diffusion coefficient and flow
rate
e
Analytical Ultracentrifugation
In analytical ultracentrifugation, analytes are separated on
the basis of their sedimentation when they experience a
centripetal force.
Usually carried out at speeds around
60,000
Analytical Ultracentrifugation
Or, described mathematically
r
N
M
r m F
s
2 2
e e = =
This is counteracted by the Boyant force (due to displacement
of solute) and the frictional force
mass of a single molecule
radius from center of spin
angular velocity
r m F
b
2
0
e =
mass of solute displaced
s f
f F =
coefficient of friction
sedimentation velocity
v
N
M
m =
0
Volume occupied by 1g of solute
Solvent
density in
g/mL
s
r Nf
M
s
=
2
) 1 (
e
v
Sedimentation
coefficient
Sedimentation Velocity Experiments
The most basic type of ultracentrifugation experiment is to
measure the rate at which the analyte moves away from the
center of rotation
What is actually measured is the movement
of the boundary between dissolved analyte
and empty buffer
s
r Nf
M
s
=
2
) 1 (
e
v
ND
RT
f =
) 1 ( v
=
D
RTs
M
r
(
= c r s
r
c
rD
r r t
c
2 2
1
e
o
o
o
o
o
o
Sedimentation Equilibrium
In sedimentation equilibrium, an equilibrium is established
between sedimentation away from the center of rotation
and diffusion towards the center of rotation.
2 / ) (
0 ,
2
0
2
) (
r r
A A
e C r C
=
o
RT
M
2
) 1 ( e v
o
=
Density Gradient Centrifugation
Density gradient is similar to equilibrium sedimentation
except there is a permanent solvent density gradient. This
sharpens the contrast between the sedimentation equilibria
of samples with the same shape, but slightly different
masses:
Meselson and Stahl, P.N.A.S., 44,
(1958), 671-682)