7 replies to this topic

[Stefff]

Hi!
I have a problem with gels cast using disposable plastic cassettes.
I've cast IEF gels (6 % containing 8M Urea/6 % Ampholytes 4-6, polymerized with 0.11 % TEMED and 0.03 % APS) and ran an IEF separation (1h at 150 V, 1h at 250 V. 15h at 350 V, 1h at 500V). After the run, the gel was detached from the cassette (see picture), resulting in no current.
I don't know, when the detachment happened, since I ran the IEF separation over night, but I stained the gel the last time and saw, that the proteins were stuck in the wells. So I think, that the incident happened early after starting the run.
I ran the separation under the same conditions before, and had great separations (several times, not just once). The problems started some weeks ago, I am using the same batch of casting cassettes as well as the same reagents as I did before. Does anybody have any suggestions?

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[ascacioc]

I do not have experience with those type of gels. However, it looks too me like your gel got dry inside. I have exactly that picture when I use the urea/acrylamide gels with high acrylamide concentration. The higher the concentration, the more dettachment I have, starting from the margins of the gel. Moreover, the older the gel the more dettachment I have. So....maybe your gels are too old. You  are actually saying that you have the same batch of gels and that before it worked perfectly, but one week ago they started not working.

[mdfenko]

the cassettes may not be holding together as they did when new. you may have to find some way of clamping the cassette closed when pouring and running.

the actual separation of the gel may be caused by excessive heating. watch the power readout early in the run.

[Stefff]

Thank you for your reply! I cast the gel immediately before running, but the acrylamide solution was not fresh, it was stored in the fridge for several weeks. So maybe the acrylamide solution was too old itself. Never had problems with that before, and it was stored in a dark bottle, but I will try it with freshly prepared solution.
The cassettes I used are disposable pre-made ones, so I don't have to clamp them for pouring.
Do you think it could be over heating? The gel itself looks good, when I take it out of the cassette, it is just partly detached. There are no bubbles in the gel itself, just between gel and cassette.

[ascacioc]

I do not think it is the acrylamide. If you cast the gel just before using it, then it cannot be that it is too old hence overdried. Then it is overheating. Do you have fresh running buffer? You can have overheating because of the buffers sometimes.

[Stefff]

Well, I am doing an IEF run, so I am working with 25 mM H₃PO₄ and 50 mM Tris base as electrolytes. And I used them before, too. I'll check the electrolytes, maybe I did something wrong while preparing them. And I try to cool the camber from the outsude. Maybe this helps, too. Thanks for your advises!!

[mdfenko]

did you allow the solutions to warm to room temperature before casting the gel?

cold solutions can cause bubbles, distortion, etc

[Stefff]

Thanks for all the help! I found the mistake, it was really just an overheating issue. I do not why though, since I ran the same gels under the same conditions before and they worked well. However, I cooled the electrophoresis chamber from the outside and limited the current - and it worked!