Our lab has currently run into some problems involving protein migration in our SDS gels, and I was wondering if anyone has experienced something similar, or could provide some insight into what might be causing our issues.
I have attached an image for you to see.
We have suddenly had an issue where when we try to separate protein samples with SDS-PAGE, our higher molecular weight proteins merge into a single line (which is also visible as a translucent "bump" going through the gel). Oddly enough in this image, this line happens a bit lower (right above the dye front). I tried using Coomassie to see if maybe it was our ladder having issues migrating, but the protein stain also showed the protein samples were running exactly how the ladder appears.
The gel will start off running normally, with good separation of our ladder (each band is visible), but then the higher weighted bands will merge back together as a single line, and will slowly overtake every other band. Additionally, these gels have been running quite a bit more slowly than normal. The image attached to this was after about 2-3 hours at 120 volts.
So far, we have remade all of our solutions, have used another labs solutions, tried changing power packs, and have tried different cassettes incase it was faulty wiring. Nothing has been working, and we are all out of ideas.
May I know your running buffer composition and pH and the recipe for your resolving and stacking gel to be able to help you properly?
Hello Gaelan,
Have you check your protein concentrations? If it was really high, try to lower it. Also, try to use an appropriate gel concentration.
Good luck,
Ahmed
Hi, if this is a new problem never happened before in your SDS page experiment try to figured out what changed in the last month. In my experience, for example, we did not known to have problem with our water distillator and all the solutions became to precipitate during the SDS page.
Did you run different samples, for example prepared in other time to figure out if the problems is some lysis buffer solution, high samples salt concentration? I not said anything about gel or running buffer because you said that you just checked out everything.
Hmmm..tough one. From what you're describing, it looks like it's something with the actual box itself (when the assembly comes together). Are you casting your own gels or using pre-made ones (and are the loading buffers, running buffers, etc etc, matched to what you are using)? Unless you're running a very long gel, 2-3 hours is a very long time..and if the current flow is perturbed, many funky things can happen, buffer exhaustion, heating, etc, especially after an hour...are your gels hot during the run? If you're having a localized electrical problem, that can result as well. Other than obvious things which you have mentioned and tried, that's the only thing I can think of....other than new cassettes, maybe a whole new apparatus?
It may be due to the pH of the reagents or quality of distilled water which is used for the preparation of solutions. secondly also check the loading protein concentration and try to load minimum quantity of protein in wells.Good luck
it sounds a bit like buffer depletion in which case try running the gel for a while then discarding the buffers and load fresh buffer and see if the proper migration continues. If so you will know where the problem lies. Alternatively cold this be a multimeric protein and the ph as the run progresses or increase in temperature as the gel runs is causing it to split into monomers which run quicker than the multimer. This would suggest that your initial denaturing conditions are not working and you would need to look in that direction
Any chance of a better pic? I don't really know what I'm looking at! And perhaps remove the pint of Guinness from behind the gel?
Can you upload a more destained gel. You can try:
1. Do not have empty well between the samples. Try loading 1X loading buffer without sample between the sample lanes.
May be if you upload better looking gel we can have some more suggestions.
Good luck.
Did you check the composition of the sample buffer and if somehow changed color? If the solutions are ok, try to re-prepare the loading buffer and run the gel less than 3 hours...
We always chill our gelsystem during long runs so the temperature is always the same during the whole run. Hope you can solve your problem.
composition of loading dye ? Glycerol concentration..............?????
The picture is not the best, but it looks like the molecular weight ladder is running well but is being displaced by the protein being loaded in the next well...If I am not mistaken...To check if the reagents are working properly you should try running only the molecular weight ladder. If this runs well then the problem is the sample itself.
I do agree with Nancy, once you run only molecular wt. marker without protein samples that is good idea.
you can load minimum to maxium quantity in one gel and varying concentration of boiling mix or glycerol into it on the other gel if you are using BioRad miniprotean system
The time that it takes to run the gel suggests that you have a much higher gel% concentration than you think or, too much resistance in the electric current conductivity (too much salts). Since you are able to load the gel I would look at the separation and not at the stacking part of the gel. I would also look at:
- Loading sample composition: As Nancy said, look at the loading sample buffer and your sample composition. Be sure you don’t have to may salts, Guanidine (precipitated with SDS and affects contiguous gels) or interfering detergents.
- Gel and Buffers percentage: Make sure that when preparing the gel you are diluting your stock solutions and not using them 10X
- Reagents: Finally, I think you should check the expire date of your reagents.
These may seem silly, but I have encountered all of them. Good luck
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