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I have a western blot troubleshooting question that I haven't been able to find the answer to in manufacturer troubleshooting guides.

As a bit of background, I was transferring 2 western blots together in the same tank (we use a BioRad tank that fits 2 transfer modules side-by-side). Normally when transferring two blots in the same tank we run for 2hrs at 20v, as the tank takes about 30 minutes to get up to 20v. However in this case after 1hr the blot was still only running at 4v. I checked the tank and the power pack temperature, and both were not noticeably hot, so I don't think the pack was struggling with high resistance. After 2h the blot was still only at 4v, and had barely transferred at all.

Upon dis-assembling the transfer module I noticed that the sponged inside all had turned yellow, however only the sponge touching the positive electrode, and only the side of the sponge facing the positive electrode.

I'm not really sure what went wrong here to be honest. The only thing that I notably did differently was that I made the transfer buffer up the day before and left it in the ice machine overnight. The transfer buffer was glycine and tris with 20% methanol, in distilled water.

Any thoughts would be appreciated.

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    $\begingroup$ What concentration of glycine in the buffer? Sound very much like your transfer buffer wasn't correctly made or you had a non-conductive layer in the western that wouldn't normally be there. It is very unusual for Voltage to not reach the set point almost immediately. $\endgroup$
    – bob1
    Jan 27, 2022 at 19:52
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    $\begingroup$ Did you check the wiring in the transfer tank? Something could be corroded or otherwise damaged to the point of breaking, allowing only weak current to pass through. Also, make sure the leads are clean (use a brass brush to remove any corrosion) and of course check that your power supply is working properly... $\endgroup$
    – MattDMo
    Jan 27, 2022 at 22:32

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Thank you for the comments. When I got back into the lab I double-checked the chemical shelf and it turns out I had put NaCl into the buffer instead of glycine! Currently soaking the sponges over the weekend to see if the yellow colour comes out...

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