Hi Stephanie,

It looks like the problem was in your sample. some of the samples may have had too much salt. try rerunning them after acetone precipitation, and resolubilize them with your sample buffer. yellowing of the bromophenol blue is usually associated with acidic pH, which impacts electrophoresis. Did any of the samples you loaded was yellow? if so, you should adjust sample pH.

Hi David,

Thanks for your answer!

I thought about salt content because of the color too but it seemed very weird to me that only some of the samples should have too much salt or a different pH, because they were treated the same.* I was also doubting this because the "blur" occured in the same position on both gels, whereas the samples were not connected anyhow. When I loaded the samples, they were blue.

* To clarify:

These are cell lyastes - I wash the cells with PBS, aspirate PBS, and scrape them in RIPA buffer. Even if a small amount of PBS was left behind, it could not have been more than a couple of µl which got diluted in 250µl RIPA buffer. I did the same for all my samples, the only difference between them is a transfected plasmid.

Still, do you think PBS could be the issue?

Some residual PBS would not be of any consequence in your sample. It is intriguing that you have a yellow front in some samples, in the same positions even though they are unrelated. This suggests something related to the equipment, rather than the samples, which normally would be pretty unlikely. To me it looks almost like something acidic is turning the bromphenol blue in the front to its acidic form, which is yellow (a colour shift due to pH change). Could there be some contaminant on the equipment (localized to the area of the lanes in question) that is counter-electrophoresing into the gel from the bottom? If this is a possibility, I would look carefully at the dry gel box components and clean them if necessary. I doubt it is the cathode wire, but you can carefully polish it with a VERY fine abrasive sanding sponge and a little pressure to remove any discolouration on the metal (be really careful to avoid doing anything that would break the wire). Try running something routine then, with all the wells exactly the same and see what happens. Sorry, but it's hard to be more specific.

I dont think it is related to your sample. It looks like somethings wrong with the equipment. There should be an alteration in the voltage part that leads to these specific wells maybe?

Are these two gels both have the same lot number? Maybe something was wrong in their production line. Try with a different lot numbered gel, or cast any gel and load the same samples that corresponded to those wells. If something wrong in your samples they should look weird no matter which well you put them into.

If you have the chance borrow someones SDS page aparatus and try there aswell? 

Do I see big air bubbles under the wonky area?

This would not explain the yellow colour but the weird running. We cast our gels ourself but we always remove big air bubbles trapped under the gel.

Thanks everyone for your suggestions!

I carefully cleaned all of the equipment now and so far, everything runds smoothly again.

I think Norbert Kartner's suggestion might have been qhat happened (counter-electrophoresing of a contaminant, because only the lower half of the gel was affected).

@ec Sener

Coincidentally I took the last gel from one box/lot and the other is from a new box, otherwise, this would also have probably been one of my first guesses.

@Victoria

I think what you mean/see is only foam from the buffer that got trapped underneath, but it's good acvice to look out for bubbles anyhow, thanks!

I agree with Victoria's comment. The air bubbles and foam trapped underneath the gel causes an uneven running of your samples. To get rid of those bubbles it helps to gently tap the gel or I also use syringes. I bend the needle of the syringe to an angle of approx. 45 to 90° and then I take up running buffer and squirt it under the gel and flush the bubbles away. 

Hello Dennis, thanks for the advice!

So I ran one "test gel" after the cleaning and two gels with my samples which all came out perfectly fine, then ran two more gels with samples and had the same issue again, this time the yellow blur only appeared on one gel, the other just had a smear/bump in the dye front.

Now that Victoria and you both pointed it out, I also get what you mean - I thought you meant the bubbles behind the gel, but now I noticed the small ones that seem to be trapped underneath. I borrowed a chamber from a neighboring lab and ran my remaining samples, ensuring no bubbles were trapped and everything was fine.

As for our chamber, I'm not 100% sure if the bubbles are the only cause, we never had this issue in our lab before and honestly nobody has ever payed attention to the bubbles. But I will definitely watch out for it in the future and warn my colleagues, perhaps it was really just very bad luck on my side that I happened to be the first person to have this problem.

Thanks everyone for your help!

I have never seen anything like this, except when there were problems casting our home-made gels.

If I saw this effect, I would send the pictures to the gel manufacturers and ask for a refund! They may well have had similar complaints!

I'm pretty sure it has nothing to do with bubbles, or dirty platinum wires, etc.

G

Hello,

Why dont you cast the gels at you laboratory?. It hardly takes 1 hour to cast a gel. 

Hi everyone, I wanted to give an update in case someone is curious how we resolved the issue.

After quite some time, the manufacturer finally got back to me and we went through everything we tried, and the support person concluded that it must be the electrode assembly that's to blame (i.e. the part we you put the gels in). In the end we also ran two other assemblies in our chamber (to exclude the chance that it's the contact on the lid of the chamber).

So we had to order a new chamber, but how everything should be fine.

Thanks everyone for your help with troubleshooting in the meantime!

@Pranav: We use gradient gels to resolve the particular proteins we want to see optimally.

Thanks for the update!

I think the manufacturers explanation is wrong, and that they had a (hopefully)  transient problem preparing those gels.

Do let us know if all is indeed OK, or what the actual problem was, if not the tank.

Geoff, I would think so too if it hadn't happened with gels from two different boxes/batches, also my colleagues and I never had these problems when we used the gels from the second batch with the other assemblies that we tried.

(Grasping straws)... but I guess the gels are prepared on large scale in multiple casting tanks before being sealed individually. So if one tank had the problem mix, and those gels were randomly distributed into the boxes of 10 that you buy....? 

That could be the case, but then we got really lucky that all the gels we ran with the borrowed electrode assemblies ever since then were always fine.

At any rate - the assembly is actually not that expensive (we paid 230€ or so). If the problems re-occur with the new one in the near future, I'd of course consider the gels as underlying cause next.

So can I tempt you to go back to the previous assembly and see if you get the same old problem? If not, as you wont be using it again, and if you haven't thrown it out in disgust, can I have it...please?

G

I'm not sure we still have the old one, I'll have to ask our technician!

Please shoot me an e-mail (stephanie.kainrath[at]ist.ac.at) to remind me to ask her on Monday when she's back :)