I was hoping someone might be able to provide some insight into what could be causing our issues with protein SDS-PAGE. We are using the BioRad mini-PROTEAN tetra cell. If anyone recognizes any of the symptoms of our issues, please share your expertise and help us diagnose the problem!

I have also attached some images for consideration.

Here is a brief summary of our issues:

- Our separation is taking significantly longer than it should; some times it can take ~1.5-2 hours @120V, we also had an instance where @110V the dye front did not migrate more than an inch after 3 hours

- gels are separating at the wrong %; for instance, we have been making 8 and 10% gels that separate as if they were 12 or 15%

- our BioRad dual color pre-stained ladder will begin to disappear around the 75kDa marker; this effect will then permeate the molecular markers above it and slowly disappear also. The weird thing is you can see a pink/blue tinge throughout that lane, so it is almost like the ladder is not migrating at a specific front, but is instead just diffusing throughout the lane

- some of our gels will also show uneven migration with one side of the gel migrating faster than the other

- the dye front runs uneven

One of the frustrating issues is that this is apparently random. Sometimes one SDS-PAGE run will work fine, other times only 1 of 4 gels might be affected by something, and they generally only show one problem when there is a problem at all.

These will then translate over into issues with our Western where there is noticeable unevenness in the protein signal especially in the middle lanes. Interestingly, when our ladder begins to disappear, our protein signals below 75kDa are completely fine, signals near 75kDa are entirely uneven and splotchy, and signals ~100kDa and above will begin to show signs of issues depending on the severity of the disappeared ladder.

The random nature of the issue has made it very difficult to trouble shoot.

These are things we have tried to troubleshoot so far:

- Remade all of our buffers

- purchased new acrylamide

- purchased pre-casted gels

- changed out our old power system for a brand new one

- tried different pre-stained ladders

- used other labs to make all of our materials

None of this has helped. One of the only promising things we have tried was using another labs SDS-PAGE equipment. These were definitely running noticeably quicker with no obvious errors. We have also used other labs equipment with our reagents and it has seemed fine.

I have narrowed the issue down to either being our power packs or our power outlets. I find it hard to believe it is the power pack as we have issues whether we use an old one, or one that is new and has only been used maybe a dozen times. I am also not sure if it might be a power supply issue as plugging another labs power pack into the same outlet showed improvement.

At this point, we are completely at a loss. If anyone has any insight, I would really appreciate it.