Constant mA vs. constant V for electophoresis, whats the difference? - (Mar/03/2008 )
Hi, I'd like to get some enlightenment on the art of electrophoresis for acrylamide gels.
Sometimes, I see people recommending running the gels at a constant V, some constant mA. What is the difference?
Also, is there a simple formula as to how long we need to run the gels and at what V/mA given a certain % gel? I run large gels for the second dimension after IEF, and a lot of times it takes me up to 8 hours to run. I wanted to run the gels overnight at a lower V/mA but I wasn't sure if that would compromise the relative patterns of the protein spots (ie. squished or expanded horizontally). And if so, what V or mA should I set if I want to run it overnight?
whether you use constant voltage or constant current mostly depends on how you were brought up.
many use constant voltage, and that is fine. the choice of voltage is pretty much gel size independent (within limits, you still want to set a maximum current or power) whereas the current you use depends on the size of the gel you are running (again, with a maximum voltage setting to prevent arcing). the setting and limits are to limit heating of the gel so that banding patterns are regular and reproducible.
in the olden days we used the canalco (later ames) cylindrical (tube) gel apparatus with power supply. the power supply only had an adjustment for current. the voltage would float (with a maximum of 500V). we would use 2-3mA/gel.
with more modern equipment you can adjust voltage, current or power (with crossover when limits are reached). i'm sure that you use constant power for ief (allowing voltage and current to float, within limits).
as one who was raised with constant current, i use that for most gels (at least, protein gels).
we run large gels. in fact, our small gel is bigger than normal (as opposed to mini) gels that most use. our small gel is 24ml before stacking and our large gel (affectionately referred to as a "blanket" by neighbors) is 63ml before stacking. we run them overnight. the small gel is run at 5mA overnight (we finish it at 15mA when we arrive in the morning). the large gel is run at 15mA overnight (finished at 50mA).
so, you can run your second dimension overnight, but limit the running constant so that it is not quite finished when you come in (you can figure it out by seeing how long it takes at one condition and adjusting the conditions from there, it is pretty linear).
so choose your constant of choice (or upbringing), set your limits and start running.
Enlightened indeed, thanks!
Our gels are 40mls before stacking (we use the Bio-Rad dodeca system), I assume your conditions are mA/gel, correct?
I'll give that a try today!