#Voltage and current to work done time full#

As discussed above, the constant-current setting is a good option for keeping the timing consistent, but may result in increased heat production. Timing will vary for this step, ranging from 45 min to 2 hours. Small gels will run closer to 100V, while large gels may approach 300V. One rule of thumb is to set your voltage at about 5-15 V per centimeter of gel. Once your proteins are on the resolving gel, you can increase the voltage. The larger the gel, the higher the voltage.This step should take about 30 minutes at 50-60V. Whether or not you use a stacking gel or other gradient acrylamide gel, it is best to start your SDS-PAGE at a low voltage or current to get the proteins lined up in the gel. General guidelines for SDS-PAGE conditionsĮach protocol and experiment is different, but there are some general guidelines for starting your SDS-PAGE optimization. Power is the product of two variables, voltage and current, so “constant” conditions are hard to define May limit the production of heat while maintaining a more consistent migration speed Migration will slow down late in the run, which may necessitate adjustments to the running time Voltage (and heat) will tend to increase later in the run, causing “smiling bands” or warped gelsĬurrent will decrease as the run progresses, which limits the production of heat May require less monitoring since the time needed to complete run will be the same across multiple gels The respective advantages and disadvantages of each setting with respect to heat are outlined below: This increase can indirectly add to the production of heat under the constant current setting since the voltage must increase proportionally.Ĭonversely, heat will remain more level under constant voltage conditions, since the increase in resistance leads to a decrease in current. Higher settings for either parameter will increase the temperature of your buffer and gel.Īs the electrolytes in your running buffer get used up, resistance naturally tends to increase. Heat production, measured in Joules, is directly proportional to the power consumed, and therefore influenced by both current and voltage. 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.

#Voltage and current to work done time full#

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. Heat is a double-edged sword when it comes to SDS-PAGE. V = I x R How does heat affect the SDS-PAGE conditions? Resistance is related to voltage and current by Ohm’s law: In Western blotting, resistance is the measure of how efficiently the ions in your SDS-PAGE buffer allow charge to “flow” through the gel. 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. 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: Using the same water analogy as above, the current is the rate that water flows through the pipe. The higher the voltage, the higher the electric “pressure” and the faster your proteins will run.Ĭurrent (I) refers to the flow of electric charge past a point in a circuit. If you think about electricity like a water tower, voltage is the water pressure created by placing the water at some height. 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. Before going to some recommended settings, here’s a quick refresher on the basics of electric circuits:

What is the difference between constant current, constant voltage, and constant power?īecause 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).

Here, we've outlined some basic principles of optimizes the first step - SDS-PAGE. It is necessary to optimize each stage independently, as each can affect the final results. 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). If you are new to Western blotting - or trying a new protocol for the first time - you’ll need to optimize the electrophoresis conditions.