I ran a gel yesterday, and for some reason the running buffer was extremely hot after one and a half hours of running the gel. I feel like this shouldn't be allowed to happen but am I right?
I was using the Bio-Rad Mini PROTEAN tetra system tank for my blot. In this system, you have a device that holds two gels in the same system. This allows you to fill the space in-between the two gels and cover the electrode in running buffer. However I was only running one gel, so I filled the whole tank with running buffer. I did this as I like to ensure my wells in my gel are submerged in running buffer before adding anything to them. However, I believe this may be the reason for heating up, I'm assuming because it increased the resistance to much. The gel also took unusually longer to run-although it was a 12% gel (i am investigating an 18kDa protein), after running for 1hr45 minutes at a higher voltage it still had not reached the bottom of the gel. As the tank was getting too hot, I had to put it in an ice tray for the last 30 minutes, and chose to stop after 2 hours of total running to prevent overheating.
Despite my worries, I carried on with the transfer onto a nitrocellulose membrane, treated it with antibodies and still got the protein bands I expected. i was running a test at the time, as the last time I ran a blot with these samples I had excessive binding. I was testing with different antibody concentrations to check whether we needed more diluted antibodies, however my control, using the exact same conditions as last time, worked fine this time. I'm not sure if the heat could have reduced protein content which could lead to my problem with banding being "fixed" for now but just because of this.
Apologies for the long answer, but my question in principle is-is it ok for running buffer to heat up during electrophoresis, and what could be potential effects on a blot if it isn't?
Not OK. If I understand what you wrote, it sounds like you did not have separate inner and outer buffer chambers. Your electrical current needs to run through the gel from one chamber to the other (same principle as a battery) and this current is what pulls your sample/protein through the gel.
If your inner tank is not separated from the outer tank by your plates, you are effectively short-circuiting your system, explaining the heat.
Even if you want to run one gel, you need 2 plates to separate the inner and outer chambers (If you did do this and I misunderstood your question, then ignore my answer).
It's not OK, most likely one of the buffers is wrong (perhaps the 10-fold running buffer concentrate was mistaken for the 1X due to mislabeling?) or there was a mistake while assembling the electrophoresis system. The increased conductivity will result in loss of resolution due to increased diffussion, which may or may not have a noticeable effect -in your case it looks like it didn't- and if the temperature is high enough, there might even be warping of the plastic components of the system, etc.
Not OK. If I understand what you wrote, it sounds like you did not have separate inner and outer buffer chambers. Your electrical current needs to run through the gel from one chamber to the other (same principle as a battery) and this current is what pulls your sample/protein through the gel.
If your inner tank is not separated from the outer tank by your plates, you are effectively short-circuiting your system, explaining the heat.
Even if you want to run one gel, you need 2 plates to separate the inner and outer chambers (If you did do this and I misunderstood your question, then ignore my answer).
Not OK. If I understand what you wrote, it sounds like you did not have separate inner and outer buffer chambers. Your electrical current needs to run through the gel from one chamber to the other (same principle as a battery) and this current is what pulls your sample/protein through the gel.
Kunal Pal, you again copy pasted Joshua Samsoondar's answer. That is not OK. Do you understand what you wrote?
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