anti-Polyglutamate chain (polyE), pAb (IN105)

Figure 1: Western blot analysis of protein polyglutamylation by anti-Polyglutamate chain (polyE), pAb (IN105) (Prod. No. AG-25B-0030).
Method: HEK-293T cells are grown in standard culture conditions, transfected with plasmids expressing the tubulin glutamylases TTLL4 and TTLL6 (J. van Dijk, et al.; Mol. Cell 2007) and are lysed in SDS sample buffer and run on a 10% SDS-PAGE. Brain tubulin was prepared following standard procedures (M. Castoldi & A.V. Popov; Protein Expr. Purif. 2003). Different C-terminally modified variants of GST-telokin were produced in bacteria as described before (K. Rogowski, et al.; Cell 2010). The proteins are transferred to a nitrocellulose membrane and detected by standard immunoblot protocol using the anti-polyE, pAb (IN105) (1:2'000) in TBS containing 0.1% Tween-20 for washing steps and 2.5% fat free milk for antibody incubation. The same samples were probed with the anti-Polyglutamylation Modification, mAb (GT335) (Prod. No. AG-20B-0020) for comparison. In the normal HEK-293T cell extracts, no tubulin detection is observed, even on high exposure. Expression of TTLL4 glutamylase does not lead to detection with anti-polyE, pAb (IN105) while glutamylation is observed with GT335. This indicates that TTLL4 generates short glutamate chains on tubulin. Expression of TTLL6 generates long glutamate chains (J. van Dijk, et al.; Mol. Cell 2007), which are detected by anti-polyE, pAb (IN105). Brain tubulin is a heterogenous mixture of different glutamylation states of tubulin (B. Edde, et al.; Science 1990) and therefore strongly detected by anti-polyE, pAb (IN105) and mAb GT335. Anti-polyE, pAb (IN105) also detects gene-encoded linear C-terminal glutamate chains. To illustrate the specificity of anti-polyE, pAb (IN105), recombinant GST-telokin proteins are included in the western blot analysis. Anti-polyE, pAb (IN105) specifically detects glutamate-chains of four and more glutamates. In contrast, GT335 cannot detect such chains (K. Rogowski, et al.; Cell 2010).
Picture courtesy of Dr. Sudarshan Gadadhar & Dr. Carsten Janke, Curie Institute, Paris