Frowning and Smiling - problems with SDS-PAGE (Oct/25/2008 )
I've been running SDS-PAGE gels for a while now, but I have a constant problem with frowning and smiling of the dye/protein front. Everything runs fine until about 1.5cm from the end (mini hoefer gels and bigger ones) the front starts to go haywire, turns yellow (obviously acidic), and when I take the gel out of the plates, the front is hardened, but this softens once I've soaked it in buffer for a while. I only run these gels at max 20mA, never more than 180V in the cold room, and it happens with Laemmli, Tricine and Bis-Tris gels. I load equal volumes in each well, fill up the unused wells with loading buffer, and make the gels fresh.
I have no idea what is wrong, and perhaps the only thing I can think of is old APS ? (I store a stock in the -70oC) or old loading buffer ?
I appreciate any comments or suggestions you could make.
fresh APS is always good
smiling and frowning is often caused by an excess of glycerol. I would make new loading buffer. this is easy enough, and will tell you quickly if that's the problem
It shouldn't be the APS, so long as the gel sets. I would suspect a buffer problem, maybe the recipes you are using are wrong or one of the components common to the buffers is decomposed. I would suspect that it is the component that is depleted by electrophoresis in the lower buffer chamber.
I also thought it was a buffer problem, but this is why I've tried 3 different SDS-PAGE gel buffer systems (Tricine, Bis-Tris and GlycIne) and they all do the same thing. I remade the acrylamide stock yesterday, and it had the same problems. I also used someone elses protein sample, equal volume in each well, and containing sucrose as opposed to glycerol. Is there a possibility that the actual gel tray containing the buffer could be faulty ? I don't see how, I clean the thing with hot water to get rid of salts etc. I also made the gels individually as opposed to a batch. I find it hard to reconcile buffer exhaustion, as I do not run them quickly and everyone elses gels that I've seen seem to run straigh to the end.
Any other ideas ?
Have you tried borrowing someone else's buffers? The only way to sort out a problem like this is to go through it systematically and change one thing at a time. If there is someone in the lab or a nearby lab who is running gels and they are working, then go and watch them run the gel/prepare the buffers etc, it may be that you have changed one component unconsciously that is affecting your experiments.
Is there a component of the buffers or loading dyes that is common to all the ones you have tried and may be off? Are you storing your loading dye properly? DTT based loading dyes need to be stored at -20 deg C.
Go back to the basic protocols and read them again carefully, there may be something that you are skipping over/not doing right in there.
I am going over my calculations with someone else, but I have tested almost everything of everyone elses except the buffers, so I'll get someone else to make them for me and then test them. I agree, one needs to go through this systematically, reagent by reagent.
Thanks for the suggestions