anti-Polyglutamate chain (polyE), pAb (IN105)
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
|Product Type||Polyclonal Antibody|
Recognizes C-terminally located linear alpha-glutamate chains of 4 and more glutamate residues.
|Purity Detail||Epitope-affinity purified.|
|Formulation||Liquid. In PBS containing 0.02% sodium azide.|
|Shipping and Handling|
|Short Term Storage||+4°C|
|Long Term Storage||-20°C|
After opening, prepare aliquots and store at -20°C.
Avoid freeze/thaw cycles.
|Use/Stability||Stable for at least 1 year after receipt when stored at -20°C.|
|Product Specification Sheet|
Microtubules are key elements of the eukaryotic cytoskeleton that dynamically assemble from heterodimers of α- and β-tubulin. Two different mechanisms can generate microtubule diversity: the expression of different α- and β-tubulin genes, referred to as tubulin isotypes, and the generation of posttranslational modifications (PTMs) on α- and β-tubulin. Tubulin PTMs include the well-known acetylation or phosphorylation, and others that have so far mostly been found on tubulin, detyrosination/tyrosination, polyglutamylation and polyglycylation. These PTMs might have evolved to specifically regulate tubulin and microtubule functions. Polyglutamylation is a PTM that occurs when secondary glutamate side chains are formed on γ-carboxyl groups of glutamate residues in a protein. Enzymes catalyzing polyglutamylation belong to the TTL-like (TTLL; Tubulin tyrosine ligase-like) family of glutamylases. Deglutamylases, the enzymes that reverse polyglutamylation, were identified within a novel family of CCPs (cytosolic carboxypeptidase). Subtle differences in polyglutamylation can be seen on diverse microtubules in different cell types. The functions of these modifications remain to be studied. However, its wide distribution strengthens the idea that it could be involved in fine-tuning a range of microtubule functions. PolyE labels centrioles as they mature, such that two foci are present throughout the cell cycle.
- Tubulin polyglutamylation stimulates spastin-mediated microtubule severing: B. Lacroix, et al.; J. Cell Biol. 189, 945 (2010)
- Microtubule detyrosination guides chromosomes during mitosis: M. Barisic, et al.; Science 348, 6236 (2015) (Supplement)
- Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning: B. Wolf, et al.; Open Bio 7, 160231 (2017)
- Alterations in the balance of tubulin glycylation and glutamylation inphotoreceptors leads to retinal degeneration: M. Bosch Grau, et al.; J. Cell. Sci. 130, 938 (2017)
- Spastin regulates VAMP7-containing vesicles trafficking in cortical neurons: C. Plaud, et al.; BBA - Molecular Basis of Disease 1863, 1666 (2017)
- iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications: J. Ou, et al.; Cell 173, 1 (2018)
- Imaging cellular ultrastructures using expansion microscopy (U-ExM): D. Gambarotto, et al.; Nat. Methods 16, 71 (2018)