There are different protocols for electrophoresis and blot considering the running conditions and it seems that everybody is doing it differently. Even the commercial kits recommend different conditions. So what is the difference between keeping constant current or voltage? Is there any difference at all or is that just some woodoo?
If you keep the current constant you run the samples in a constant electric field - always at the same speed. Since the high mobility ions (like chloride) are replaced by bigger (glycin anions for example) the resistance goes up. To keep the current constant this means the voltage goes up as well, which produces more heat in the end, what can make problems with the gel. Too much heat can cause smiling of bands, uneven gels and in some cases even cracked plates. It can also cause a not as nice seperation of larger proteins (for SDS gels). Against these problems you can cool the gels either with a cooled buffer systems or simple with an ice pack against the gel.
Running at constant voltage means that the field is not constant over time (it gets weaker, the longer the blot runs due to Ohm`s law) so your gel is running slower in the end but not producing as much heat. The slower migration in the end can also cause difficulties with the separation of larger proteins.
If you keep the current constant you run the samples in a constant electric field - always at the same speed. Since the high mobility ions (like chloride) are replaced by bigger (glycin anions for example) the resistance goes up. To keep the current constant this means the voltage goes up as well, which produces more heat in the end, what can make problems with the gel. Too much heat can cause smiling of bands, uneven gels and in some cases even cracked plates. It can also cause a not as nice seperation of larger proteins (for SDS gels). Against these problems you can cool the gels either with a cooled buffer systems or simple with an ice pack against the gel.
Running at constant voltage means that the field is not constant over time (it gets weaker, the longer the blot runs due to Ohm`s law) so your gel is running slower in the end but not producing as much heat. The slower migration in the end can also cause difficulties with the separation of larger proteins.
Constant Amps or Volts are a matter of convenience and performance. If you e.g. want your SDS PAGE gel to run as fast as possible, use constant Amps (e.g. 25mA per mini gel) and limit the Voltage (e.g. 250V for a mini gel). This will ensure your gel runs in slowly (70V or so) when the protein enter the gel and hit the separating gel (good for best possible performance) and run fast with high watts later in the separation, keeping it nice and fast (remember, you can not stop diffusion in SDS PAGE, so short runs equals sharp bands). It does get hot, but before it is too hot you end the run, easy. Constant Voltage is easiest to calculate if you have time and want to fit it round your other activities (e.g. 2 h at 150V, starting for 10 min at 80V or so, or over night at 40V). The longer the run the more diffusion...but might not be visible or critical in any case. Other programs optimise the run, but need programming or you changing the voltage, that is basically it...Smiling....load all lanes with loading buffer or some sample if it "salty" or something, avoids it...
It's Ohm's law. The resistance will increase during the run.
If you would like to have a constant separation speed, use constant current (which requires a constant cooling at 15 °C), if you can live with a separation speed which gets constantly slowly, work with constant voltage.
Beware: don’t cool below 15 °C, SDS can crystallize at lower temperatures.
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