Has anyone ever tried Phospho-SIRT1(Ser27)&/or(Ser47) from Cell Signalling Technology (reactivity for humans) for WB on mouse samples? Or does anyone know an available antibody for SIRT1 that reacts with mouse? There is an SIRT1 activity ELISA but this is expensive for only 100 tests. Whereas, if the Human Ab works (as SIRT1 is highly conserved) on mouse then this will be much cheaper for the number of samples we currently have.
Looking at the Phosphosite database, there is no sequence similarity at the Ser27 site:
http://www.phosphosite.org/proteinAction.do?id=11108&showAllSites=true
The Ser47 site looks similar between the two species, so the antibody might work to detect the phosphorylated murine protein. This paper seems to provide evidence:
http://onlinelibrary.wiley.com/doi/10.1111/j.1471-4159.2011.07184.x/full
Have you considered performing 2D gel electrophoresis with subsequent 2D Western Blotting? If a validated antibody against your phosphoform of interest is not available, then IEF may provide better resolution than standard SDS-PAGE. Conceptually, you could use one antibody for both the non-phospho and phosphoforms, provided that the antibody was raised against a common epitope. Moreover, 2D techniques may enable you to confirm polypeptide fragment identity by mass spec. Additional confirmation of identity by immunoaffinity recognition may be provided by dephosphorylation to show coalescence with the non-phospho-target.
Just to let everyone know who answered (many thanks!) Phospho-SIRT1(Ser27)&/or(Ser47) from Cell Signalling Technology (reactivity for humans) works perfectly on mouse skeletal muscle cell samples!
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