SDS gradient gel problems

[pizzapop]

Hi all, recently I ordered some gradient gels from BioRad, and I noticed that when I am running samples the dye smears and forms weird shape (like an upside down smile) especially at the higher gradient conc., is this normal for gradient gels or because of my running conditions. I use 1X Tris-Glycine running buffer w/ SDS (pH not adjusted), cooled, and run my gels in the cold room. I usually set my voltage to 200V and run for 1h. Should I perhaps lower the voltage?

Thanks, any help and suggestiosn would be appreciated

[danfive]

This happens sometimes, usually due to large amounts of proteins loaded on gel, while lower amounts would run straight. You have smiling bands (if you want to search) anyway, I deal with it by lowering the Voltage (135V), extending the time(+30min).

If the cold isn't essential I'd take the electrophoresis out, it affects the pH, but more importantly it affects SDS solubility, if your SDS precipitates then the run is compromised.

Usually SDS-PAGE won't benefit from cold conditions, the SDS is covering your protein, so degradation should be inhibited.

[pizzapop]

thanks for your suggestion! the reason i pre-chill the running buffer and ran the gel in the cold room is that I am afraid that my gel will heat up since i need to run it for more than a few hours and I heard keeping the running buffer cool will improve the band resolution. When I take out the gel, it feels like its around 35 - 40 degrees C, do you think at that temperature will affect band quality? thanks!

[danfive]

Originally Posted by pizzapop

thanks for your suggestion! the reason i pre-chill the running buffer and ran the gel in the cold room is that I am afraid that my gel will heat up since i need to run it for more than a few hours and I heard keeping the running buffer cool will improve the band resolution. When I take out the gel, it feels like its around 35 - 40 degrees C, do you think at that temperature will affect band quality? thanks!

If your run is 2 hours or less I wouldn't worry about the heat. I'd change to room temperature. But excess protein on the gels seems to cause a lot of resistance and that causes a lot of heat. Lessening the protein on gels should solve the problem.

If the electrophoresis apparatus and buffer get too hot, (you should monitor) again this is due to too much protein (and/or too much salts), then lowering the voltage will help (low voltage would be 100V, with 135 being medium range).