Laemmli Buffer Problems - my lanes collapse on themselves (May/19/2010 )
When running western blots of samples which have been boiled in laemmli buffer, occasionally my bands will collapse into a thin line, rather than a full lane, making it impossible to make sense of the gel. What am I doing wrong?
Let me see if I understand your description correctly: instead of fat, separated bands, you get one thin, run-together one? Or are they separate and thin?
Maybe your (precast?) gel is old, or something on the plates is preventing the gel from adhering well. You could be getting slow leakage around the edge of the gel.
kmwalters on May 19 2010, 03:17 PM said:
DNA in the sample maybe? I've been doing boil preps for westerns with bacterial cultures and have noticed if the samples I load are too concentrated, it looks like there is a string down the middle of the lanes that the proteins stick to that causes a distortion in the band. Instead of being a nice uniform band, it turns almost into a "W" shape or something along those lines. I don't know for sure what causes that, but assumed it may be DNA from the prep, or some other cellular debris. If I sonicate the pellets instead of boiling, I can get a higher concentration without the distortion. I've read that the sonication breaks up the DNA a lot better than the boiling does. I'll see if I can find an image of one of my gels or westerns to show the distorted bands.
Here is a band from western... doing a coomassie of a gel basically looks the same, but with all bands like that, not just the one.