Phospho-ATF-2 coming up different sizes - (Jun/25/2012 )

Hi,

I am doing a western blot for the phosphorylated version of nuclear transcription factor ATF-2 (Alternative Transcription Factor-2), I am using an antibody from Cell Signalling Technologies (http://www.cellsigna...ducts/9221.html) fo anti-phospho-ATF-2.

The antibody datasheet says that the protein should form a band at around 70kDa, however tha band on our blot seems to be around 45kDa instead, which is quite a large difference (photo attached, the smaller band is the COX IV loading control)). I know that phosphorylation can make proteins appear larger on blots due to the higher negative charge afforded by the phosphates, but not to the tune of an extra 25 kDa surely, and why would it be SMALLER. The antibody was supposed to be specific for the phosphorylated version of the protein.

What is making things more complicated is reading other datasheets of antibodies against ATF-2 from other manufacturers, and their datasheets say that they expect bands at around 54-60 kDa (http://www.abcam.com...body-ab809.html) (http://www.abcam.com...dy-ab11908.html) (these are unphosphorylated versions, so the discrepency could be down to the phosphorylation) But then I come across another antibody saying that the expected band is at 52 kDa but the observed band is at 37 kDa. Which greatly confuses me. (http://www.abcam.com...dy-ab60661.html)

Even more confusing is reports in literature giving sizes of ATF-2 from anywhere between 50kDa to 80kDa depending on the paper.


anyway sorry for all the waffle, does anyone have any good ideas why the apparent size of ATF-2 could be changing so much in the various applications, and why it could be so different in my experiment?

If you look carefully, you can see a faint doublet band on your upside-down blot at 60-70kDa. This is likely p-ATF2. The other band is likely some other protein that you don't care about. Try running a positive control lysate (i.e. the same cells on the datasheet 293 +/- UV or anisomycin) next to your samples. It will also be useful to do a positive control treatment (UV or anisomycin) to your cells.