what is making my current decrease in SDS-PAGE? - SDS-PAGE Current and Resistance (Jun/22/2008 )

Help!

My proteins won't run down their lanes because my current is too low. I've used fresh running buffer everytime, but my current begins at 50 mA but then steadily declines to 0.1 ~1 mA ( I HAVE NO IDEA WHY). I'm using a BioRad voltage pack 1000, and this has never been a problem for me until now? Does anyone know why my current is decreasing, or likewise why my resistance is increasing???

-desperate technician who needs results soon

-milesperhour-

QUOTE (milesperhour @ Jun 22 2008, 01:39 PM)

Are you keeping the voltage constant at too low value (unknowingly)? That may do it. As the gel runs, amp continues to come down anyways (unless made constant, and in that case, voltage would go up).

You may get some tips here: http://search.vadlo.com/b/q?sn=158621799&a...rrent&rel=0

..

-cellcounter-

Resistance is increasing as the electrolytes are used up from the solution. Try running your gels at constant Amperage, as this will automatically adjust the volts to suit.

-bob1-

QUOTE (bob1 @ Jun 22 2008, 08:26 PM)

Resistance is increasing as the electrolytes are used up from the solution. Try running your gels at constant Amperage, as this will automatically adjust the volts to suit.

I tried. The resistance increases, so the machine shuts down because the voltage runs too high.

-milesperhour-

Is your running buffer fresh? or is it made up correctly. Maybe if its been used too much or the components are off then you'll affect the electrolyte balance. I would always run at constant voltage anyway, I find the current fluctuates a little- I guessing as the electrolytes in the buffer being used up. I'm guessing though that the running buffer maybe your problem. If you have another power pack try that and see if it makes a difference or make sure you tank is cleaned out thoroughly- rinse with dH2O to make sure there isn't any crystals of buffer or something that maybe interferring.
Also check that your gel is all assembled and there's enough buffer in the chamber as that sometimes affects the way a gel runs.
Hope one of these suggestions works for you!

Lost

-lost in the lab-

Forgot to say, check the leads from the powerpack to the tank, they sometimes break or get damaged which can lead to less current and problems running gels.

-bob1-

thanks guys! the biorad rep told me to ensure separation between the inner chamber and outer chamber. u need a closed circuit in order for separation of charges and hence a proper current to flow. My current still decreased, but it held steady at around ~19mA as opposed to 0.1 mA.

milesperhour

-milesperhour-