Hi, I'm getting troubles with the transfer of my western blot. I'm performing a semi-dry transfer (BioRad transblot SD) with constant amperage (0.25 A) but then the voltage is increasing to 45V, which should not be more than 25V. Any idea what could be going wrong? Thanks in advance.
P.S. The transfer buffer is 20% methanol, I'm checking regularly that the filter papers don't dry and the membrane is PVDF
Dear Enrique Andres
This paper can help you : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3456489/
Dear Enrique
during transfer and buffer electrophoresis, your buffer is broken down by passage of current, I.e. electrolysis. Thus during the course of transfer your buffer inonic composition will change and this will affect the voltage in real time according to Ohms law
The important thing is that you keep the current constant: Current provides transfer impetus and keeps the buffers from over heating if constant
Hi!
If you used current volume of WB buffer 2-3 times it have to change its compound (evaporation, electrolisis). It increases buffer resistancy. Use fresh buffer and check its compound again.
Best regards,
Tatiana
Depending on the buffer concentration and duration of running the voltage will increase in order to maintain the constant current to 25A. This is because ions present in the buffer move towards positive charge and makes less ion in negative charge area. In order to compensate to maintain Constant current, the voltage increases.
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