Protein transfer, high resistance causing loss in voltage? - (Mar/03/2006 )
Hello everyone. I had a question. I am using an older Bio-Rad power supply that is supposed to go up to 500V. When I get out my multimeter and test it the readings are correct. I used it for my gel electrophoresis and it went to the proper voltage but now when I am doing my Transfer to the membrane, I can only get it to go up to 50 v. Is my apparatus creating too much resistance? I didn't know if there was a common problem that caused this. I am using a Bio-Rad Mini PROTEAN III for both applications.
It's quite possible that the gel sandwich has a high resistance, though have you checked what the limiting variable is? You might be restricting the supply to a capped value for power or current.
Besides, why are you going to use such a high voltage for transfer any way? For the semi-dry transfers that I've performed in the past I used 16 V for about 30 minutes. Even with my new Invitrogen Blot Module I still only need 30 V for 1 hour. 500 V will end up boiling your gel. Think hot an agarose gel gets after 45 minutes at 150 V.
Besides, why are you going to use such a high voltage for transfer any way? For the semi-dry transfers that I've performed in the past I used 16 V for about 30 minutes. Even with my new Invitrogen Blot Module I still only need 30 V for 1 hour. 500 V will end up boiling your gel. Think hot an agarose gel gets after 45 minutes at 150 V.
Thanks for the reply, turns out that the power supply didn't have enough amps to raise th voltage high enough due to the higher resistance., but i found on that did. I was only going to run it at 100V for an hour. that was what Bio-rad's mannual said, in past labs I did it at 65v for hour but i am sticking to the Bio-rad spec for now. Also isn't 30 V slow for only 1 hour, i thought 30 to 35 V for for overnight transfers?