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-   -   Voltage vs current in Tricine SDS PAGE-ˇˇ (http://www.molecularstation.com/forum/sds-page-gel-electrophoresis-forum/72171-voltage-vs-current-tricine-sds-page-%A1%A1.html)

Autophagator 01-05-2010 05:30 PM

Voltage vs current in Tricine SDS PAGE-ˇˇ
 
Dear all: A member of this forum 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 product between the voltage and the current (Power) improve (or decrease) the resolution of the PAGE? If you know the right answer: thanks in advance

Ghanem 11-02-2010 01:20 AM

Re: Voltage vs current in Tricine SDS PAGE-ˇˇ
 
I think that it would be better to make a big gel to see the difference.

BioBM 11-02-2010 09:41 PM

Re: Voltage vs current in Tricine SDS PAGE-ˇˇ
 
You are correct in that setting the voltage effectively determines the current given a specific gel and buffer (in other words, given a material with fixed resistance). Gel boxes don't have any variable resistors that would additionally adjust the current beyond that.

...and Ghanem is right, if you can't get resolution with a minigel, just use a larger gel. Larger gel = more runtime with a given voltage = proportionally more separation.

Cheers,
-C


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