duplet bands in western blot - advice (Oct/06/2005 )

Hi,

I am using a purified antibody to detect about 50KDa protein. I always get duplet bands or one very strong band together with smear on top of that band I am looking for (see the attachment). this antibody has been used for sereval people, including few in my lab. For them, it seems only the lower band is detectable.

Any ideas why?

I am using 15 well, while others using 10 well gels. Does the well width make difference?

Thank you.

Ann

what makes the difference may be the voltage at which you run your gel. For getting an idea of the quality of migration, you can check how look like the protein mw marker, and stain your memebrane with ponceau and see how like the proteins...
if all looks sharp, maybe you have a post translation modified protein and detect the different forms?
fred

The well width shouldn't make a difference. What are you looking at? Is it a cell surface protein. If so the bands could suggest different glycosylated forms.

I see the same thing from time to time and have always assumed it was due to the massive amount of protein added to the well; it seems to be a function of overloading...you see the same sort of short halo above a DNA band if you overload too

I never assumed it was a different protein, if everything else looks good (controls, etc)

I would guess the reason you see it more than your colleagues, in a 15-well comb there will be narrower lanes and you would see overloading more easily, compared to a 10-well comb in the same width gel?

Anyways, I could totally be wrong, but that is what I think it is.

Thanks for the reply.

this is a nucleus protein and the ones I am showing you is the phosphorylated form. I am sure it is not because of the different degree of phosphorylation. In some cases, I am seeing a smear on the top of the smily bands (see attachment)

I do not think I am loading a lot of proteins. For other antibodies, I am loading 3-5 times more proteins, yet never see this kind of situation.

If it isn't a phosphorylation Then... What about a glycosylation event or ubiquitinylation?

I've seen published western blots with bands smearing like yours. I've heard that ubiquitinylated proteins are sometimes smeary on western due to different numbers of ubiquitin groups added.

Personally, I would try rebloting your membrane with an anti-Ubiquitin or something. Who knows maybe the phosphorylated protein is targeted for destruction or something? The IkB inhibitory subunit of NFkB is regulated in a similar manner.

Moutainman