Semi dry blotting - calculating the current

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Fri Jun 19 13:33:04 EST 1998


Hi all!

The electrophoresis safety debate now hopefully has alerted all 
electricity experts here, hasn't it?

My question/poll? is on current setting in semi dry blotting.

A constant current of 0.8mA/cm2 (makes approx 4 to 5 volts) is run 
for 2 or 3 hours. (Says the lab manual and has been done so for 
generations here, so don't fuss with it...)

* One fraction (myself) in the lab calculates the absolute current 
from the dimensions of the filter paper (usually 12.5 x 25 cm; thus 
360 mA), while the
* other fraction (the rest of the lab) calculates the current by the 
dimensions of the nitrocellulose membrane used.
The latter usually is considerably smaller, this way only half or 2/3 
of the current is applied that a full size calculation would reqire.

It seems that both calculations are resulting in transferred 
proteins, so who's really right i.e. what is the correct way of doing 
the calculation? 
In my opinion, the dimensions of the wet paper define a conducting 
plane. Since a density of current is defined, one must look at the 
whole conducting plane, not only at the membrane.

But what are the practical consequences? Sometimes I'm afraid 
to loose protein through the nitrocellulose since I have the higher 
current (and the higer electric field strength) for the same time 
compared to the other calculating method, where the current is 
clearly lower.

Anyway: why is current used for electroblotting and not voltage as in 
gel electrophoresis? The protein transfer should be driven by the 
electric field, not by charges moving through the buffer (and 
generated resp. destroyed by electrolysis of buffer component, 
caused by the current). 
Is current setting just used by convenience since it's quite tricky 
to adjust a constant voltage of about 4 volts (yielding an electric 
field of about 10V/cm) while the current easily may be adjusted 
accurately to less than 1%? (The reason could be that the voltage 
meter doesn't resolve well in this range, it's just one digit)

The constant current setting in my opinion could cause problems due 
to the uncontrolled / not reproducible warming up of the assembly. 
(This leads to an increase in electric field strength because warm 
buffer has a higher resistance than a cold buffer. For keeping the 
current constant, the supply increases the voltage. This probably 
will shorten the transfer time necessary or move proteins across the 
membrane).

Is there anybody who's able to give an answer ?


Wolfgang



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usual disclaimers apply * This message is RNAse free - please don't touch!
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Wolfgang Schechinger         
University of Tuebingen, Germany
email: wgschech at med.uni-tuebingen.de * wwWait: http://www.medizin.uni-tuebingen.de/~wgschech/start.htm
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