Western Blot smudge - (Jul/10/2006 )
We obtained the above western blot last week and there are three issues I would like to troubleshoot on it, and they are numbered.
1. The huge black "smudge" seen has never been present even on the worst of our westerns. Could this be due to overloading a lane with massive amounts of protein?
2. Why are these bands inverted?
3. This is a lane of recombinant protein. The lane was greatly overloaded (correct target is 5 ug, actual loading was 100 ug). Could this have caused the smudge seen in (1)?
A few months ago I had found a website that showed dozens of problematic western blot pictures. It was essentially a troubleshooting website and I recall seeing a picture there that was just like what we came up with last week. Does anyone know the website I'm talking about?
I think the site you're looking for is the SDS-PAGE "Hall of Shame" found here. I don't know if there's one for blots as well!
Based on your picture, their pictures and my own mishaps, it looks to me as if your gel was poorly made as well as overloaded (100 ug would certainly do it, as well as distort everything beside it). The fact that the recombinant protein is not in one fat blob like [1] but rather elongated into the lane is weird to me since I've never seen that sort of thing outside a full lysate stain, and certainly not probed on a blot (maybe someone else has seen this?)--but that my be a bad gel issue. As for those negative bands I'm not sure, but they may just be bubbles if the blot membrane wasn't in full contact with with the gel.
Anyhow, good luck on future blots!
hi i need a help in my western
i work on MT1-MMP and there are no bands shown in the membrane
what can i do??
did you pour your gel by yourself: are you sure the pockets were evenly polymerised and no air bubbles in it
are you sure your protein extract is homogenous and no bigger particles in it which might run strangly (then perhaps additional sonification might be neccessary)
I once got "negative bans" when my blot was overdeveloped and to much background so the prestained marker showed as white bands on the film....
Actually, these are premade Bio-Rad gels. The first one we did was perhaps the cleanest western blot we had ever seen, then the above pictured blot came out...and it looks terrible. So hopefully this was a fluke and if it is true that this was a poor gel, then I'll keep my fingers crossed that the rest of the Bio-Rad gels aren't bad, as well, assuming that is the issue.
Anyway, thanks for the website. The Hall of Shame is exactly what I was looking for. While it's not a western blot website, the erroroneous techniques apply, since a WB is essentially just an image of SDS-PAGE.
Thanks for the help!
Definitely use 1-5% of the amt of sample for the next run.
It is possible that ion concentration in your sample was too high. try dilute your sample or dialyse it before loading see if that will help.