Electrophoresis
A tool for separating specific biomolecules
from a mixture
(A key step in most biotechnology
applications)
Electrophoresis:
movement of charged particles through a
substance when an electric field is applied.
http://www.dnalc.org/ddnalc/resou
rces/electrophoresis.html
The Gel Substance
• Agarose (from seaweed)
• Polyacrylamide
– Powders that gel when boiled in liquid and
cooled to room temp.
The Buffer
• Ions in solution
– Conduct the current from the electrodes
through the gel
– Maintain pH so that biomolecules keep their
original charge
THE ELECTRIC FIELD
Parallel Electrodes
Electric field (direction of
conventional current)
Electron flow
- cathode
+ anode
typical gels
-
-
+
+
Movement of biomolecules
DNA, RNA, Proteins
• Strong electric field → large force moves
molecules faster
– For nucleic acids (DNA and RNA), average
voltages range from ~80-200 Volts DC
• The more concentrated the gel matrix,
the slower the migration
Goal:
• Maximize the separation of molecules
of different molecular weights
• Minimize changes in electrolyte
conductivity
• Don’t overheat (melt) the gel
Physical features of biomolecules affect
their ability to be separated from each
other:
• Size (molecular weight)
• net charge
• Conformation (shape)
Size and Net Charge
DNA
-
-
-
-
-
-
-
-
-
-
-
-
-
External Phosphates give
DNA a net
Negative charge.
Longer molecule means
a lot more atoms and
a few more negative
charges.
As length increases,
charge/mass ratio
Decreases.
Larger molecules go slower
Conformation (shape)
DNA can be supercoiled - goes faster than
when it is linear
Proteins can take on many different 3-D
shapes – These are denatured in the gel so
that they are all essentially linear.
Who can resist a little Physics?:
Force diagram
Q+
Ffrictio
n
+ anode
- cathode
FElectric
∑F = FE + Ff = ma = 0
Biomolecules migrate at Terminal velocity
A little Chemistry:
- cathode
Water is
H20 → H+ + OH-
e2H+ + 2e-→
H2(gas)
+ anode
Electrolysis
e2H2O→
O2(gas) + 4H
+
+ 4e-
• The generated amount of hydrogen is twice the
amount of oxygen.
• Both are proportional to the total electrical charge that
was sent through the water.
Electrophoresis LAB
1. Characterize physical features of dye
molecules (relative size, charge, relative
amount)
2. Determine whether dyes are pure (only 1
color) or complex (more than one color)
3. Identify the 2 dyes in an unknown
mixture
These are some of the individual colors
in your dyes
Blue #1
792.85 g/mol
Fluorescein
376.27 g/mol
Crystal Violet
408.0 g/mol
Yellow #5
452.38 g/mol
Red #40
496.42 g/ mol
Safranin
350.85 g.mol
Terminology
• Pure dye has 1 color
• Complex dye has more than one color in
it.
• Your unknown is made of 2 dyes (may be
complex, pure or one of each)