Semi dry blotting - calculating the current

Frank O. Fackelmayer fof1 at chclu.chemie.uni-konstanz.de
Sat Jun 20 04:43:25 EST 1998


Wolfgang Schechinger wrote:
> 
> 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.

Actually, I don´t see the problem. I thought everyone sticks to the semi-dry
protocols that say "cut the membrane and papers to exactly the size of the
gel". That´s the first thing I tell my students when we do semi-dry blotting.
Consequently, there is no difference in the current to apply. 

> 
> 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.

agreed

> 
> 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.

The capacity of membranes used for blotting is very high, so the risk of
losing protein is very low. I would recommend to blot only for 90min. Unless
you have a protein >100kd, that´s usually sufficient.

> 
> 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

agreed

> 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)

I think its by convention and convenience. 

> 
> 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
> 
> -----
> usual disclaimers apply * This message is RNAse free - please don't touch!
> -----
> 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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