Identify the band in western blot - (Mar/19/2010 )

Hi. I have a polyclonal antibody which apparently detected specific band in cancer cell lines. However, after a treatment a few days ago, another band present, indicating the overexpression of unknown protein after my treatment. Is there any method for me to identify the protein? Preferably method with low cost..

Any suggestion is much appreciated.

dcch on Mar 19 2010, 04:06 PM said:

Well as it is polyclonal u never know if its another protein which is cross-reacting with your primary or a modification of your own product!!!
Now if the antibody is specific and yet there is band.. which boils down to it being your product of interest with some modification .. say truncation, deamidation... can be anything... in which case running a 2d wud be a good idea!!

Hope it helps..
Best luck!!!

I agree with the previous answer. If this band is not normally present in your western blot but only appears after treatment, your protein of interest is most likely being modified. Is the new band close in size to the original band? Is it higher or lower? The most common modification is phosphorylation which commonly (but not always) results in an upshifted band. You can split your sample after treatment and subject one to phospatase treatment to see if the new band disappears.

rkay447 on Mar 21 2010, 03:40 AM said:

You can see phosphorylation, sumoylation, ubiquitination... a picture would help us

Yeah, I'd bet on phosphorylation. Perhaps you know if your protein would be a phospho-target of some kind?

But you can try a proteasome inhibitor like MG-132 to see if your protein accumulates for Ub or try running the protein sequence through a sumo site identifier program like SUMOplot or SUMOsp. If you do think its SUMO, I'd run the other way. Ub and Smt both have specific ab you can buy that aren't too expensive.

You can use mass spectroscopy to identify the band specifically. A machine with an orbi-trap will work well for phosphorylated samples.