Actually is not just "smiling", it´s kinda "roller coaster" effect, with ups and downs. I´m attaching a pic

I really do not know your specific run conditions and sample solubility in terms of salt concentrations amongst other things but heating may cause it. I have preferred running gels in a cassete that allows for a closed cooling system of refrigerated recycled water. it worked for me. This allows you to choose the temperature that the gels are run. if you the heating effect is eliminated and it persists pls let me know

Thanks, Oluyinka, but the gels are kept cold during the run. Must be somethig else...

so sorry to hear that. perhaps you may try to make sure that your proteins are COMPLETELY soluble and you may run in using lower power to increase longer run times. Again precast commercial gels may have the problem if stored. Freshly made lab gels gave better results in my experience. Am sorry if i cldn't be of better help. all the best.

Don't feel sorry at all. Thanks for commenting.

Best.

Use fresh sample buffer, mix with the sample and heat immediately and cold in -20 C subsequent heating. Ok, thereafter, run at constant low voltage ie 80-100V for mini gel and 165-180V for medium size gel.

ot could be a buffer thing, i find that happens when i have samples that have different buffers or pH or molarities. It also happens when the running buffer leaks out of the container.

Thank you all for the comments. Please, note that these are Phos-Tag containing gels, not just regular acrylamide gels for SDS-PAGE. I posted the question here because this is a phosphoproteomics forum and hoped someone may have used this reagent.

Thanks, anyway.

I don't know if you have solved this already but I have used the Phostag system and have found that if you use a low constant current it helps with the smiling effect. There are a couple of papers to this effect. I have gone down to 5mA and you don't see that effect. The only thing is that it takes a while to run the gel. Somewhere around 15mA seems to work alright.

http://onlinelibrary.wiley.com/doi/10.1002/pmic.200900020/pdf

http://www.nature.com/protocolexchange/protocols/501

HI Jackie,

thanks for the suggestion. We will definetively try lowering the current.

Best.

I wonder if lower current helped your issue. We have seen roller coaster effects but also smearing, almost as if there's some discontinuity in the gel itself. Any ideas would be very much appreciated.

I don't know whether you guys already solved the problem.

I have also seen the smiling and smear during phostag separation. I solved this issue by clean the sample with methanol/chloroform precipitation and resolubilized in lamelli buffer. I also found if I load the gel with hamilton syringe, the residual contamination inside the syringe could change the separation dramatically. So Now I always use clean gel loading tip to load the sample.

Thanks Yongsheng, haven´t tried that. Could you provide a detailed protocol?

We´ll give it a try.

http://drummond.openwetware.org/ChlorMethExtraction.html

this is a good one.

Good luck!

Sorry, forgot to mention: I did not dry the pellet by vacum dry, but air dry for 5 min on the bench. If the pellet is too dry, then it is very hard to resolubilized again.

Thank you, Yongsheng.

Hi Jose,

though it's 3 years passed from the last post, was wondering if you could solve the problem? I have noticed when I make the gel, even before loading samples etc. when I look at the gel I see there is a smiling boundary in the middle of the  gel, it's like there is sth in phostag or manganese which make the acrylamide gel funny. Just was wondering if you solve the issue, and how?

thanks