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IgG - unspecific band (Dec/01/2010 )


An unspecific band appear at 55kDa on my western blot, i suppose it is IgG.
my protein of interest is at 50kDa!
How can i get rid of IgG band?


Protein A or G chromatography.


does the band appear all the way across the membrane? if so then you may be experiencing an artifact that shows up when using aged reducing agent (presumably keratins from dust).


I assume this is a western blot of an IP. You have a couple good options. One, many companies are now selling "light-chain specific secondary antibodies" that are specifically designed for western blot after IP and will not bind the heavy chain. I tend to like Jackson ImmunoResearch and have had very good success with their products:

Otherwise, you can use protein A-HRP as your secondary. It isn't as sensitive as most other secondaries but it will only bind antibodies in the native conformation. This means that it can't bind the denatured heavy or light chain that was used in the IP and run on the gel.