Power setting for electrophoresis - (Jun/21/2005 )
I always have been confused by which settings are accurate for different types of electrophoresis. Could anyone help clarify the concepts behind it? Thanks.
I was told by my advisor to follow the rules for the following, but he simply failed to explain to me why.
Set constant voltage at 130V (for minigel), set lower current --> so that voltage will be run at the set limit
Set constant voltage at 100V (for BioRad's protean II module), set lower current --> so that voltage will be run at the set limit
Set contant current at 200mA (for BioRad's protean II module), set lower voltage --> so that current will be run at the set limit
1. Are the current settings accurate?
2. Why is it that blotting different from agarose electrophoresis/SDS-PAGE in terms of setting constant current vs voltage?
Many thanks in advance!!
for agarose gel electrophoresis, i use 100 to 130v and there's no problem. due to the fact the gel and running buffer are both in 0.5xTAE (for me) it's not a problem to get constant voltage.
For protein electrophoresis, a great protein specialist told me that SDS page should not start over 80v. Hence he advised me to start at 80V constant, chek the amperage (17mA in mini protean system, 1 gel of 0,75mm spacing), and go to constat amerage. Voltage gonna slowely increase but intensity is the same ensuring an homogenous migration...
For Agarose gel electroforesis it depends on the dimension of gel. It should be voltage 3-6 V/cm (measured as a distance between electrodes). I have a box of 17.5 cm, and i run at 80V. Nevertheless, it depends also on your aim. If you want just to check the product of PCR on gel, you can put 100V ( but pay attention, some low-melting agarose could not support high heating), if you want to divide many bands, better to apply lower voltage.
For SDS PAGE it depends of the apparat construction, always better to check the device manual. Earlier, we had Hoeffer Mighty Small, we have run it on 80 mA, and with obligatory water cooling; And very often, when somebody was too impatient, and gave a bit more current, the glasses was splited by overheating.
Now we have a new one Invitrogen , for the same gel dimensions, and we run it at 200mA, without any cooling, and it is genial, run time is about 30 min
Unless the application is critical, I just run my agarose gels at constant 100Volts and make sure the amerage does not exceede .15Amp.
I run a hoefer mini-gel, 25mA thru the stack, 35mA thru the resolving. Voltage starts off at about 60, rising to 150 towards the end of the run..takes about 2 hrs. I use no cooling and have not had any cracked plates. One dude in the lab is always cranking up his voltage and cracking plates... piss me off.
I blots at constant volts 100 volts... about 125 amps, rising to 200amps by the end of the run.. I use cooling and there is no problem.
Judging from your replies, it seems to me that agarose and SDS-PAGE/blotting should be run according to Vol and Amp, respectively.
Sorry if this is a dumb question, but I am just wondering why it isn't vice versa (as initially set Amp and Vol for SDS-PAGE and agarose electrophoresis, respectively).
The reason why I'm asking this is that my colleague ALWAYS set his power setting to be at 130V and 120mA - no matter running agarose gel or SDS-PAGE gel; and (although he had never cracked his glasses or encountered over-heating problems) he claims power settings for these two types of electrophoresis simply don't matter at all. I'm just curious about the correct theory behind all this. Thanks again!!
Because it works silly. Thats what science is all about... doing what works.