changing colour of front dye in sds-page - (Sep/23/2009 )

Hi,

I have a problem running sds-page on mini gels (hoefer system). I usually run at 100 V in cold room. Everything looks fine until the front dye reaches ca. 1/4 from the bottom of gel. Then bands start to distort and front dye changes into yellow (pH change?). I checked all my buffers for recipes - all fine. Checked pH - fine. I have no idea what can be the reason. Is it possible that the buffer gets depleted? I thought that they should design the system, so that there's enough buffer for a mini gel.

Please HELP!

kris

I use Hoeffer mini-gels for all my gels and have never had this problem. The colour change is almost certainly a pH effect, probably acidification of the gel somehow. The buffers do deplete as the gel is running and this will result in some pH change, but this should not be enough to change the pH in the gel significantly.

I would check the acrylamide and buffers used for making the gels.

The discontinuous nature of the buffer system is depleted -- the glycine ion front has run off the gel.

ok, so how can I solve this? Do I really have to change buffer in the upper chamber during electrophoresis? Or is there any other way? Do you think I could use for example 1,5x or 2x buffer? How could it affect the whole process?

this could be happening because of the buffer that your sample is in (prior to addition of the laemmli sample buffer). one or more of the components seems to have caught up to the dye front. if you let the gel continue to run it may straighten itself out.

mdfenko on Sep 24 2009, 09:11 PM said:

oh, I do let it run, but it won't straighten out. and we are using two different buffers, and they both give the same result.

kristoff on Sep 24 2009, 06:03 PM said:

what components do your buffers have in common? one of them may be the culprit.

as for homebrew's suggestion, this can happen if the buffer is made too dilute. are you sure that your electrode buffer(s) are prepared properly?

mdfenko on Sep 25 2009, 04:46 PM said:

buffer composition- checked it twice, it's the standard sds-page buffer, 1x
as for the components of the buffers: only sodium chloride is common, the other components differ.

Hi Kristoff:
just curious if you ever managed to resolve this problem? I'm having exactly the same problem (dye front going yellow in last cm or 2 of Hoefer thin minigel) and finding it difficult to troubleshoot. Clearly the pH is changing in the dye front but for the life of me I can't figure out why. I am quite sure that the samples I am loading are not to blame because the colour of the front in the lane containing molecular weight marker (to which I add 1x sample buffer to equalize the volume in all wells) is as likely to turn yellow as my cell lysate sample. After I add mercaptoethanol to my sample buffer the pH is 7.4 - could this be a problem? (I'm adding 128ul beta-ME to 1ml of 5X SB).
Anyway, I'm only beginning to actively troubleshoot this problem but if I find anything I'll repost.

Hey Kristoff and TheBalrog,

At the moment I am having the same problems with my SDS-PAGE gells, which I cast myself. My problem is not only that the dye front changes yellow but is also stops being strait which is a real problem. Has one of you guys an idee on how to solve this?


Thanks!

Elco Backus