This section provides an overview of the steps involved in performing electrophoresis, such as system setup, running conditions, selecting power supplies, gel disassembly and storage.
Related Topics: Protein Electrophoresis Methods, Protein Electrophoresis Equipment, Protein Electrophoresis Sample Preparation, Protein Electrophoresis Reagent Selection and Preparation, and Protein Detection and Analysis.
System setup involves placing the gels in the tank, filling the tank with running buffer, loading the samples and protein standards, and programming the power supply.
Regulated direct current (DC) power supplies allow control over every electrophoresis mode (constant voltage, current, or power). The choice of which electrical parameter to control is almost a matter of preference.
In a PAGE separation, the gel containing the protein sample is placed in the electrophoresis chamber, between two electrodes. The driving force behind the separation is the voltage (V, in volts) applied across the electrodes. This leads to a current flow (I, in amperes) through the gel, which has an intrinsic resistance (R, in ohms). Ohms's law describes the mutual dependence of these three parameters:
I = V/R or V = IR or R = V/I
The applied voltage and current are determined by the user and the power supply settings; the resistance is inherent in the system and is determined by the ionic strength of the buffer, the conductivity of the gel, etc.
The power (P, in watts) consumed by an electrical current element is equal to the product of the voltage and current:
P = VI = I2R = V2/R
The strength of the electric field E (V/cm) applied between the two electrodes is an important parameter in electrophoresis, because it exerts a force on electrically charged objects like proteins and determines their migration rate:
E = V/d
Most vertical electrophoresis chambers are operated at field strength of 10–20 V/cm for 1mm thick polyacrylamide gels.
The electric field strength (E, in V/cm) that can be generated between the electrodes is limited by the heat that is inevitably produced during electrophoresis. This Joule heating can lead to band distortion, increased diffusion, and protein denaturation when not efficiently removed from the system. The amount of Joule heating that occurs depends on the conductivity of the buffer used, the magnitude of the applied field, and the total resistance within the system.
The heat generated (Joule heating) is proportional to the power consumed (P):
Heat = P/4.18 cal/sec
Understanding the relationships between power, voltage, current, resistance, and heat is central to understanding the factors that influence the efficiency and efficacy of electrophoresis. The optimum condition is to run at the highest electric field strength possible within the heat dissipation capabilities of the system.
During an electrophoretic separation using the Ornstein-Davis and Laemmli systems, the running buffer warms as a result of Joule heating. The increase in temperature may lead to inconsistent field strength and separation and may cause the buffer to lose its buffering capacity of the gel to melt or become distorted. Under normal running conditions, the running buffer absorbs most of the heat that is generated, but during extended runs or high-power conditions, active buffer cooling is required to prevent uncontrolled temperature increases.
The following variables also change the resistance of the system and, therefore, also affect separation efficiency and current and voltage readings:
- Alterations to buffer composition; that is, addition of SDS or changes in ion concentration due to addition of acid or base to adjust the pH of a buffer
- Gel pH, ionic strength, and percentage of acrylamide
- Number of gels (current increases as the number of gels increases)
- Volume of buffer (current increases when volume increases)
- Transfer temperature (current increases when temperature increases)
- Gel length (increasing gel length demands higher voltage settings to increase field strength accordingly)
- Gel thickness (increasing gel width or thickness at identical gel length leads to higher current; voltage must be kept unchanged)
Power supplies that are used for electrophoresis hold one parameter constant (either voltage, current, or power). The PowerPac™ HC and PowerPac™ Universal power supplies also have an automatic crossover capability that allows the power supply to switch over to a variable parameter if a set output limit is reached. This helps prevent damage to the electrophoresis cell. Select a PowerPac power supply that is best for your application based on the following considerations.
The resistance, however, does not remain constant during a run:
- In continuous buffer systems (for example, those used for blotting or DNA separation), resistance declines with increasing temperature caused by Joule heating
- In discontinuous systems, such as the Ornstein-Davis (native) and Laemmli (SDS-PAGE) systems, resistance also changes as discontinuous buffer ion fronts move through the gel; in SDS-PAGE, resistance increases as the run progresses. Depending on the buffer and which electrical parameter is held constant, the Joule heating of the gel may increase or decrease over the period of the run
Separations Under Constant Voltage
If the voltage is held constant throughout a separation, the current and power (heat) decrease as the resistance increases. This leads to increased run times, which allow the proteins more time to diffuse, but this appears to be offset by the temperature-dependent increase in diffusion rate of the constant current mode. Separations using constant voltage are often preferred because a single voltage is specified for each gel type that is independent of the number of gels being run.
Separations Under Constant Current
If the current is held constant during a run, the voltage, power, and consequently the heat of the gel chamber increase during the run. Constant current conditions, as a rule, result in shorter but hotter runs than do constant voltage runs.
Separations Under Constant Power
Holding the power constant minimizes the risk of overheating.
Electrophoresis cells require different power settings with different buffer systems. The values presented are guidelines — conditions should be optimized for every application. However, in every case, the gel should be run until the dye front reaches the bottom of the gel. Use the recommended power supply settings for your electrophoresis system and method.
Use external cooling during long, unsupervised runs. Temperature-controlled runs often yield more uniform and reproducible results.
For best results:
- Increase run times for gradient gels and decrease them as needed for low molecular weight proteins
- If needed for your application, allow the sample to stack using field strength of 5–10 V/cm gel length for the first 10 min of your run. Then continue with the maximum voltage recommended in the instruction manual of the electrophoresis system
- If using multiple cells and constant voltage, use the same voltage for multiple cells as you would for one cell. The current drawn by the power supply double with two — compared to one — cells. Set the current limit high enough to permit this additive function. Also be sure to use a power supply that can accommodate this additive current
Remove the gel cassette and open it according to the manufacturer's instructions. Before handling the gel, wet your gloves with water or buffer to keep the gel from sticking and to minimize the risk of tearing. Sometimes it is also helpful to lift one edge of the gel with a spatula.
Stain, blot, or process the gel as soon as possible to maintain the resolution achieved during electrophoresis and to keep the gel from drying out. For long-term storage, either dry stained gels in a 10% glycerol solution (storage at 4°C) or with the GelAir™ drying system, which allows gels to dry between two sheets of cellophane within 60 min. This yields clear, publication-quality gels ideal for densitometry.
This tutorial describes the features of Bio-Rad's PowerPac family of power supplies and compares their specifications. It also offers information about parameter selection, common error messages, and available PowerPac accessories. Important safety information is also provided.