i´m measuring by WB the level of GluA1 phosphorylated specificly in Ser 845 residue. Total GluA1 antibody shown a band at 100kDa, as been previously described, and other around 140kDa . But when i try to detect its phosphorylated form, I observed in hippocampus two band: 100kDa, and 140kDa; but in cortex i only detect 140kDa band.
It is posible that a phosphorylation can induce a shift in 40kDa, in a specific brain region???
And second, if I interested in report p-GluA1/ GluA1 levels, against which band i should do it????
a 40kda real shift sure that not! ;) an apparent shift why not...
phosphorylation often is associated to a shift in the mobility so you get a band with a slightly different mobility than the reported one. Depending on the gel concentration this will go to another apparent molecular weight, and maybe this is 100 -> 140 in your conditions.
If you want prove that is the phosphorylated band there are few ways: the most straigforward is to de-phosphorylate and check that goes away: incubate your native, non-denatured extract, with a phosphatase -typically AP-. AP does not work in sample buffer nor "aggressive" extraction buffers, so you need to have native, non-denatured protein and use the appropriate buffer.
Another way is to use an anti-phosphoGluA1 antibody or even better, pull it down in a collumn with anti-GluA1 and they prove it with the anti-GluA1 antibody and a generic anti-phospho-serine antibody. Note that there are many other ways, you can even send it to MS, but it's kind of overshooting...
Once you have confirmed that is the phosphorylated band, just do the ratio but you need to use the same antibody for both (so the anti-GluA1 antibody). People usually reports the phosho/total, not phospho/non-phospho so you don't end dividing by zero or something close to zero when all phosphorylated.
Hi, David Ramonet. Thanks for your comments. It's was really helpful
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