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Voltage issues during transfer

Western blotting voltage power supply

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#1 GeHe

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Posted 25 April 2012 - 06:01 AM

The last time I was running a WB, I encoutered some problems. Running the gel went as usual, with no apparent problems. But when I was about to start the transfer, I could not get my desired voltage (100V, 2hrs). I set the the power supply unit (PSU) to 100V (constant Voltage) and pressed run. The psu almost immediately switched over to constant ampere, and the voltage would not increase beyond 43V. I switched tanks and lid with the same result. Even removed the transfer sandwich, but nothing changed. Also tried a different psu.
On the second psu there is a lcd screen where it displays the different parameters during the run. In my case it showed 55V, 500mA, 24W. I let it run for some time and the voltage slowly started climbing. After approx. 1 hr, it was 81V, 500mA and 34W.

Can anyone give me some pionters as to what may be the problem here? I use Blotting buffer: [20 % (v/v) methanol, 192 mM glycine, 25 mM Tris, 0.02 % (w/v) SDS] which I prepare as 10X Tris-Glycine stock. I did not pH adjust, however, I have used this same buffer in a previous transfer, without these problems.

My limited knowledge about electricity tells me that for some reason the psu can not establish the wanted difference in charge between the two poles/sides in the tank, even when it's pouring in 500mA. Maybe there is a "small" short circuit somewhere, causing the current to flow through it rather than generating the voltage?

Thanks..

#2 casandra

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Posted 25 April 2012 - 07:10 AM

The last time I was running a WB, I encoutered some problems. Running the gel went as usual, with no apparent problems. But when I was about to start the transfer, I could not get my desired voltage (100V, 2hrs). I set the the power supply unit (PSU) to 100V (constant Voltage) and pressed run. The psu almost immediately switched over to constant ampere, and the voltage would not increase beyond 43V. I switched tanks and lid with the same result. Even removed the transfer sandwich, but nothing changed. Also tried a different psu.
On the second psu there is a lcd screen where it displays the different parameters during the run. In my case it showed 55V, 500mA, 24W. I let it run for some time and the voltage slowly started climbing. After approx. 1 hr, it was 81V, 500mA and 34W.

Can anyone give me some pionters as to what may be the problem here? I use Blotting buffer: [20 % (v/v) methanol, 192 mM glycine, 25 mM Tris, 0.02 % (w/v) SDS] which I prepare as 10X Tris-Glycine stock. I did not pH adjust, however, I have used this same buffer in a previous transfer, without these problems.

My limited knowledge about electricity tells me that for some reason the psu can not establish the wanted difference in charge between the two poles/sides in the tank, even when it's pouring in 500mA. Maybe there is a "small" short circuit somewhere, causing the current to flow through it rather than generating the voltage?

Thanks..

but you used two different power pacs, right.....so the probability that both are malfunctioning at the same time is unlikely. Did you prepare fresh 1X buffer from the same 10x buffer which worked before?
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#3 GeHe

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Posted 25 April 2012 - 10:38 PM

Two different power packs, and prepared from the same 10x buffer, yes.

#4 mhr

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Posted 23 May 2012 - 03:37 PM

Is it possible that your transfer apparatus is causing the switch? We usually run our transfers at 280-460mAmps, which I believe is quite a low voltage. Maybe your apparatus is shutting down due to over-capacity?

#5 shyam700

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Posted 31 May 2012 - 09:02 PM

if You are transferring on PVDF membrane use and your gel thickness is 0.75-1mm range use constant current 50 mA per gel for two hrs.

I do the same ways and get good transfer.

Edited by shyam700, 31 May 2012 - 09:02 PM.


#6 Arun Kumaran

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Posted 01 June 2012 - 06:27 AM

For transfer to PVDF membrane, I use 20V for 1 hour. Keeping the current or voltage as constant is good for running gel. But for transfer to membrane, keep the voltage constant (V) since current (mA) may changes based on the resistivity during transferring. Once you set up the voltage, note down the initial current and after one hour note down the current. You can see the reduction of current (mA) from initial. This proves that your proteins are transferred to membrane.





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