voltage/amper problem in western blot - Is there a link between volts (or mAm)and resolution? (Jan/12/2010 )
A member of this forum (mdfenko..thanks) suggest me to use Tricine SDS-PAGE to separate proteins of low weight. I want to solve two proteins that differ in 2 Kd (14 and 16kd respectively), but I cant get the resolution with my minigels (12%spacer+ 15%resolving): always appear one band instead of two separated bands. I have checked all the buffers and its OK (I think..)
Now, re-reading the original paper (Schagger and von Jagow) says that the voltage AND the current must be fixed, for my gels the initial voltage must be 30V then 90V, AND the initial current may be as high as 80mA. I usually run at 30+90V and 400mA max current! So, I use Power (W) greater than the paper suggests and I still cant separate my proteins. But I dont understand, and correct me If I wrong: if you set the Voltage in the power supply, the Resistance offered by the buffer and gel will fix the current automatically, or at least that says the Ohm´s law if I remember well..
And a another question, can the voltage (or power) improve or decrease the resolution of the PAGE?
Thanks in advance.
what they are telling you to do is set the voltage and limit the current. when the current reaches the set limit the power supply will switch from constant voltage to constant current. then voltage will start to go down (as will power, w=va).
too much power can cause distortion of the gel and may reduce resolution.
but, as i said in the other topic, i think you are using too high percentage of acrylamide. your proteins are near the upper limit for the percentage you are using and the resolution will suffer.
by the way, make sure your crosslinker ratio is correct (s & vj use 49.5%T, 3%C).