Hello, I am a doctoral student in Brazil. I need to perform the transfer to check for fibronectin immunodetection (250 kDa). Our transfer buffer was heating up too much; I even changed the ice every 30 minutes and placed the transfer tank in a bowl with ice. Even so, after 2 hours of transfer (90 V), the buffer was boiling! To avoid damaging the equipment, we fixed the amperage (we monitored the variation, it started at 0.80 and at the end of 2 hours of transfer it was at 1.5 mA). I am transferring for two hours at 0.80 mA with the voltage unrestricted and the buffer did not boil. How much amperage should I set to transfer two membranes, 1.5M (10% acrylamide), overnight?
There's also another option... the transfer doesn't need to be overnight, however, when we tried transferring at 90V for 2.5 hours, the buffer heated up too much. The last few times we did 76V for 6 hours and only then were we able to transfer high-molecular-weight proteins.
Thanks
Kindly check this, I hope it will help. Good Luck. Pena, Louis. (2020). Re: What is appropriate voltage settings for western blot transfer step?. Retrieved from: https://www.researchgate.net/post/What-is-appropriate-voltage-settings-for-western-blot-transfer-step/5f15cf0895d3c60c4f520d35/citation/download.
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