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Crazy looking BN-PAGE gel - No, really. (Apr/30/2010 )

Dear scientists, I'm just running BN-PAGE for the first time and my buffer front is looking absolutely crazy with the amount it migrates varying by about 50% across the gel. I am running it in 0.02% coomassie G at 150V then changing the buffer to 0.002% when it gets a third/half of the way down and switching to 250V. It didn't seem that there was anything wrong at that point but at the end there was. As far as I'm aware there is no problem with the power pack and gel chamber since our SDS gels run fine in there. My best guess is that while pipetting out the old buffer I've made contact with/squished the gel case (this is all Invitrogen equipment) and this has somehow created air bubbles or disturbed the electric field subsequently leading to the problems.

Firstly, does this sound likely, because I didn't anticipate it causing a problem; secondly, how do other people do this exchange and have they seen similar problems and thirdly... is there another answer.

Many thanks,
IC

-Coomb Raider-

picture?

do you cast your own gels?

if so, do you stack on the apparatus?

if so, did you clean up all the spillage?

if not, check that the electrode is not partially covered by stacking gel.

or...

are you running the gel on the same bench that you run sds-page?

if not, is the apparatus properly leveled?

-mdfenko-

These were precast 3-12% gels from Invitrogen. I'm pretty sure the gel tank is level enough even though I'm running it in the fridge so the location is different. Am working on a photo, but will need to borrow a camera from someone

I'm running a fresh gel and made sure when I exchanged the solution I didn't touch the cassette at all. Still the same problem, however I realize that I didn't describe it correctly. The original buffer front is straight enough, but the trailing edge of the dark blue buffer, ie the leading edge of the light blue buffer is the line that's going wrong.

Again everything looked fine while changing buffer.

My new pet theory is that this is because I used tris not bis-tris. Could a wandering pH cause a problem like this?

-Coomb Raider-

when you change to it, do you flush the wells with the new buffer?

just pouring the buffer into the tank may not completely remove the old buffer from the wells and, especially since most pour from one side, you may be creating a sort of side-to-side buffer gradient.

-mdfenko-

Towards the end of the second run, the whole thing just ground to a halt as far as I could tell. After the buffer change I left it running for 1.5 hours at 250V and a further 3 hours at 150V and while the dark blue splodge in the centre and left of the gel didn't seem to move at all, the right edge did keep going very slowly, sloping off through the higher acrylamide.

I'm taking this as a sign it is a buffer problem and that by the time the bands have run half way the pH is drifting more alkaline and negative things are getting protonated, couldn't find the pKa of coomassie anywhere though, so maybe this isn't the answer. I will also make sure I flush the wells in future, the way the front tailed off seemed suspiciously as you predicted, thanks mdfenko

-Coomb Raider-

Seem to have sorted the issue. I changed three things 1) Using Bis-tris in buffers 2) By making sure I washed the wells when exchanging buffers at half time 3) Completely removing 0.02% coomassie and adding 0.002% rather than removing 90% of the original and adding plain. Now everything runs as it's supposed to. Of these I expect number 1 is the most important, but I will continue to do all three just in case.

Now all I need to do is detect a protein.....

Thanks to all for their input.

-Coomb Raider-