Electrophoretic Theory

Appendix A: Electrophoretic Theory

In This Section
Voltage, Current and Power; Interative Effects on Gel
Electrophoresis
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Electrophoretic Parameters205
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Appendix A: Electrophoretic Theory
Voltage, Current and Power: Interactive Effects on Gel Electrophoresis

Introduction

Ohm’s Law

Electrophoresis is defined as the movement of ions andcharged macromolecules through a medium when an
electric current is applied. Agarose and polyacrylamide are
the primary stabilizing media used in the electrophoresisof macromolecules.

Current (I)=Voltage (V)/Resistance (R)Macromolecules are separated through the matrix basedon size, charge distribution and structure. Proteins willseparate through the matrix based on size, structure andcharge. In general, nucleic acids migrate through a gel
based on size, with little influence from base compositionor sequence.
Two equations are relevant to the use of power supplies
for electrophoresis of macromolecules: Ohm’s Law and theSecond Law of electrophoresis. These two laws and theinteractions of these parameters (watts, volts, current)
are critical to understanding electrophoresis.

Ohm’s Law states that current is directly proportional tothe voltage and is inversely proportional to the resistance.
Resistance of the system is determined by the buffers
used, the type and configurations of the gels being run,and the total volume of all the gels being run.
Second Law
Watts (W)=Current (I) x Voltage (V)The Second Law states that power or watts (a measure ofthe heat produced) is equal to the product of the current
and voltage. Since V=I x R, this can also be written as
Watts=I2 x R.

Electrophoretic Theory
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Electrophoretic Parameters

If a break occurs in the system, such as a damaged leador electrode or a buffer leak, the resistance of the gel
will be greatly increased. This will cause a large increasein wattage and voltage resulting in the generation ofexcessive heat. It is even possible for the system to get
hot enough to boil, or start the apparatus to scorch orburn.
Constant current

In a vertical system when wattage is held constant,the velocity of the samples will decrease because thecurrent, which is in part carried by the DNA, decreases tocompensate for the increase in voltage. The generation ofheat will remain uniform.

0151001050

507 056Time (hours)

0

1

2

34

Volts
Watts
mA

50

12

34

When voltage is set constant, current and wattage will
decrease as the resistance increases, resulting in adecrease of heat and DNA migration.
Since the heat generated will decrease, the margin ofsafety will increase over the length of the run. If a problem
develops and the resistance increases dramatically, thecurrent and wattage will fall since the voltage cannot
increase. Even if the apparatus fails, the worst that is likely
to happen is that the resistance will increase so much thatthe power supply will not be able to compensate, and it will
shut off.
Constant voltage
200
150
100
50
0Reference1.

7 0
56Time (hours)

0123

470
60
50
4030
20
107 0
56Time (hours)

Electrophoretic Theory

20150

Volts
Watts
mA

10Constant voltage

Volts & Watts

25200mA

Contant wattage

15100
50Since voltage and current vary over time at a constantwattage, it is not possible to predict mobility of samples
from the calculation of watt-hours.

20150

Constant wattage

If the current should decrease disproportionately (from a buffer
problem, a buffer leak or a hardware problem), the power supply
will increase the voltage to compensate.

25200mA

There are advantages and disadvantages for setting
each of the critical parameters as the limiting factor
in electrophoresis. Sequencing gels are usually run atconstant wattage to maintain a uniform temperature.
Agarose and acrylamide gels for protein and DNA separation
are run at constant voltage or constant current.

When the current is held constant, the samples will migrate
at a constant rate. Voltage and wattage will increase as
the resistance increases, resulting in an increase in heatgeneration during the run.

mA

Introduction

Constant current

Volts & Watts

During electrophoresis, one of the parameters is held constantand the other two are allowed to vary as the resistance ofthe electrophoretic system changes. In vertical systems,
the resistance of the gel increases as highly conductive ionslike Cl are electrophoresed out of the gel. As these ions are
removed from the gel, the current is carried by less conductiveions like glycine, borate, acetate, etc. Under normal conditions
in horizontal systems, there is little change in resistance.
However, with high voltage or extended runs in horizontal
systems, resistance can decrease.

Volts & Watts

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Volts
Watts
mA

Rickwood, D. and Hames, B.D., Gel Electrophoresis of Nucleic Acids: A
Practical Approach, IRL Press Limited, 1982.

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Notes

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