If you are new to Western blotting — or trying a new protocol for the first time — you’ll need to optimize the electrophoresis conditions.
There are two times during a Western blot when an electric current is applied: during the initial “running” step (SDS-PAGE) and during the transfer step (sometimes called the “blotting” step). It is necessary to optimize each stage independently, as each can affect the final results. Here, we've outlined some basic principles of optimizes the first step — SDS-PAGE.
Because the conditions of the gel, buffer, and sample can change during the electrophoresis steps, most modern power packs offer a variety of options for maintaining constant voltage, constant current (amps), and constant power (watts). Before going to some recommended settings, here’s a quick refresher on the basics of electric circuits:
Voltage (V) — the difference in electrical potentials between two charges — is the primary parameter for defining the speed that your protein will move through a gel during SDS-PAGE. If you think about electricity like a water tower, voltage is the water pressure created by placing the water at some height. The higher the voltage, the higher the electric “pressure” and the faster your proteins will run.
Current (I) refers to the flow of electric charge past a point in a circuit. Using the same water analogy as above, the current is the rate that water flows through the pipe.
Power (P) — generally defined as work done per unit of time — is simply equal to the voltage multiplied by the current, which can be written as such:
P = I x V
One additional parameter to consider is the resistance (R), which (as the name implies) is a measure of how difficult it is for the charge to pass through a conductor. In Western blotting, resistance is the measure of how efficiently the ions in your SDS-PAGE buffer allow charge to “flow” through the gel.
Resistance is related to voltage and current by Ohm’s law:
V = I x R
Heat is a double-edged sword when it comes to SDS-PAGE.
On the one hand, some heat is beneficial for assisting in the denaturing of proteins that may not have undergone the full reaction during sample prep.
Too much heat, however, will cause acrylamide gels to expand, which can cause bands to run unevenly (sometimes referred to “smiling” bands) or, worse yet, make the gel unworkable for the transfer step.
Heat production, measured in Joules, is directly proportional to the power consumed, and therefore influenced by both current and voltage. Higher settings for either parameter will increase the temperature of your buffer and gel.
As the electrolytes in your running buffer get used up, resistance naturally tends to increase. This increase can indirectly add to the production of heat under the constant current setting since the voltage must increase proportionally.
Conversely, heat will remain more level under constant voltage conditions, since the increase in resistance leads to a decrease in current.
The respective advantages and disadvantages of each setting with respect to heat are outlined below:
Advantage | Disadvantage | |
Constant Current | May require less monitoring since the time needed to complete run will be the same across multiple gels | Voltage (and heat) will tend to increase later in the run, causing “smiling bands” or warped gels |
Constant Voltage | Current will decrease as the run progresses, which limits the production of heat | Migration will slow down late in the run, which may necessitate adjustments to the running time |
Constant Power | May limit the production of heat while maintaining a more consistent migration speed | Power is the product of two variables, voltage and current, so “constant” conditions are hard to define |
Each protocol and experiment is different, but there are some general guidelines for starting your SDS-PAGE optimization.