Ghost bands on SDS gel - Problem with blur bands (May/12/2008 )
I was just wondering what could be the cause for some 'ghost' bands on SDS gels. Recently, the SDS gels I've been running have clearer bands for higher molecular weight proteins and very blur bands for lower molecular weight proteins (See attachment). I think it is a problem with the gel and not with the protein samples since even the markers appear to have this problem and also, previous gels did not have this problem.
I prepare my own SDS gels with 12% acrylamide for the resolving part and run them at 180V for 1 h.
Any comments welcome
I would say this is normal. Low-weigth proteins tend to difuse more in the gel. You are using stacking gel, right?
Are you sure that the gels you used before had the same composition? You can try using a higher-percentage gel to get sharper bands, but you will have to prolong the run and the higher weigth proteins won't separate as nicely. Still, for pretty pictures, higher percentage is usually the better option.
or run a gradient gel to keep the bands as sharp as possible.
Thanks! Hopefully changing the gel will resolve the problem.
Simon: I was pretty sure that I ran similar samples before with a better resolution for the lower molecular weight bands (see Second gel attachment). I didn't change any of the gel running conditions though so it could be something to do with the sample (?). But then again, the markers are blurrer in the first gel but not in the second.
For sharp bands:
1. Use a 4%stacking gel and run it at a lower V, then after samples enter to the resolving gel use a higher V.
2. Run gel in cold, better if put you put the chamber in the refrigerator.
I figured out the problem with my gels was due to the staining and destaining solutions I was using. I used pre-stained protein markers to follow when the ghost banding appeared and found that after running the gel the marker was still clear but after staining and destaining it became blur. I made up fresh stain and destain solutions and the problem is gone now (see attachment, Top: new stain and destain solutions, bottom: old batch of stain and destain solutions).
The gel resolution is still not great at 12% (I ran the first gel before all the suggestions) but at least I'm not wondering where all the proteins have gone!
Thanks for all the troubleshooting hints and tips
For future reference:
Merlav: What's the lower V that you run it at before running it at higher V?