BN-PAGE crooked dye front?
I am running a BN-PAGE and for some reason sometimes the dye front isn’t straight. The gel seems to run more slowly in the 2nd & 3rd and 8th & 9th lanes (of a ten well gel) than all the others, kind of like an upside-down “W” (see attached picture). This does not always happen and sometime only happens on one side. This is not sample specific. It can happen even if I run the gel with only one sample in the middle lane (or one sample in the 2nd/3rd/8th/9th lanes) with all other lanes empty.
I run the samples at 4°C in a cold room (I have also tried on ice, but that did not help). I run at 100V the entire time since any less and the samples won’t enter the gel. I fill both cathode and anode buffers to the top of the gel running apparatus and the cathode buffer is not leaking into the anode buffer. I am using a 4-12% acrylamide gradient gel (ExpressPlus™ PAGE, GenScript, M41210). My cathode buffer consists of 50 mM Bis-Tris, 50 mM Tricine, 0.02% Coomassie G250, pH 6.8. I switch to the same buffer with 0.002% Coomassie G250 1/3 of the way through the run. My anode buffer consists of 50 mM Bis-Tris, pH 6.8. I pH all of my buffers at 4°C since I run my gels in a cold room.
I suspected this is due to uneven heating, I have measured the temperature within the apparatus and it is about 16.5°C at the top of the gel and 15°C at the bottom. I have tried stirring the cathode buffer inside the apparatus, but that did not help (and could damage the apparatus). I’m not sure what else to do about this.
***NOTE that I do not usually put a stir fly in the cathode chamber of the apparatus even though it is present in the pictures.
I run a few BN-PAGE gels 3 years ago, so I'm definitely not an expert, but I don't remember running any buffer loaded with coomasie G250. The protocol I used only called for G250 in the loading dye that's added to the protein sample before loading the gel ... so I basically run it almost like an SDS-PAGE gel. It worked fine and the added bonus is that apart from the moving dye front, you also see your protein bands as they migrate through the gel ... as far as I can remember I had no problems with the shape of the dye front.
If they were home made gels I would say that there is a problem in the polimerization of the acrylamide. As Antonio, I have never used Comassie inthe running buffer, only in the loading buffer. I cannot see the adventage of using it in the running buffer.
Are you sure that the gels are ok, are not out of date? or , Are there any problems with that batch?, have they been well stored? Are there other people using them with good performance in the lab?
You could check also the electoforesis electrodes. When you run another kind of gels, the apparatus works well? Check it preparing a common home-made SDS-PAGE.
Hope it helps!
Judging from your helpful pictures, my suspicion is that the level of buffer in the inner buffer tank is too low - and current does not pass through the parts of the gel that are not covered. Note that the end wells do seem to have less buffer than the central wells and they must all be filled and the inner chamber level must be higher than the inner plate.
There are also a lot of bubbles: are you sure that the SDS is the correct concentration in the buffer? It may be that the bubbles are effectively displacing the buffer from the inner chamber.
Also, I always assemble the gel chamber and fill with buffer to check that there are no leaks before I put the assembly into the tank. And I always check that the buffer level is correct during the run. If the level falls, I stop the run and top up the inner chamber.
In PAGE without SDS proteins move toward the positive electrode on the base of their own charge. Some proteins are neutral therefore cannot move and precipitate in the gel. I suggest to add 0,1% SDS in your running buffer and to run at room temperature your page checking eventually watt in the power supply that should be less than 10 w.
The gels are precast and I am contacting the company because I think it was the gel. I ran gels from another lot and I did not have this problem (see attached picture).
My guess is that the "problem" gels might not fit you apparatus as perfectly as they need to. But no harm contacting the company - you might get a refund!!
We have a Bio-Rad Mini Protean apparatus and they said it would fit (I even contacted them to make sure of it), but the cassette is a bit thicker than the precast gels we have used in the past. I hope it was just a bad batch of gels. I haven't been able to find any precast gradient gels appropriate for native page that fit our apparatus, and I would prefer to avoid casting them myself if possible since that might waste even more time.
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