Hello everyone,
I am running a western blot for pSTAT3 (Santa Cruz antibody, host: mouse). recently (three days ago) I started to see a strange, aspecific band, which is equal in all lanes and size corresponds to heavy chain. My western is on total extracts from cells, not on IP. I did not change any conditions. Secondary antibody is of course anti-mouse. I thought it might be something in my samples (they were all from one experiment) but the second time I included as a control a sample from a previous experiment which gave only the correct band before, but now also shows the aspecific one. Has anyone ever seen anything like this or maybe has any ideas how to explain this?
Kris
Well, I have to worry, because it appeared all of a sudden, I've been using this antibody for quite some time already and it never gave this band and also this new band is very strong, so strong it sort of diminishes the strength of pSTAT3 antibody.
could be a dimer or an aggregate? Try to add 2ul/sample after boiling of b-mercaptoethanol before loading a new gel
I assume that the band is around 55kDa. Since STAT is above 90kDa it cannot be aggregate. A wild guess (if you are working with epithelial cells) keratins???? They are usually not very well solubilized, so the amount in the lysate vary depending on lysing procedure / time.
Hope your lysis buffer / reagents are not contaminated with antibodies. Is total STAT antibody / any other antibody with the same secondary behaving the same
You can try using Clean blot IP or some light chain specific secondary to confirm whether it is antibody or not..
Good luck
Krzysztof,
giving space to the immagination....
are you using protease inhibitors in the lysis buffer? Some of them are recombinant proteins purify by tag-affinity chromatography. If you are using a polyclonal antibody produced with the protein having the same tag, can be possible obtain tag reactivity in WB...may be..
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