anti-α-Tubulin (acetylated), mAb (TEU318)
Method: HEK-293T cells grown in standard culture conditions, transfected with plasmids expressing the tubulin acetyl transferase α-TAT1 (or MEC17; J.S. Akella, et al.; Nature 2010; T. Shida, et al.; PNAS 2010) and are lysed in Laemmli sample buffer. Brain tubulin was prepared following standard procedures (M. Castoldi & A.V. Popov; Protein Expr. Purif. 2003). Samples are run on a 10% SDS-PAGE and proteins are transferred to a nitrocellulose membrane and detected by standard immuno blot protocol using anti-α-Tubulin (acetylated), mAb (TEU318) (1:2’000) in TBS containing 0.1% Tween-20 for washing steps and 2.5% fat free milk for antibody incubation. In normal HEK cells, acetylated α-tubulin is detected, although faintly. Expression of α-TAT1 (or MEC17) acetyltransferase leads to enhanced acetylation of tubulin. Brain tubulin is highly acetylated and therefore is strongly detected.
Picture courtesy of Dr. Sudarshan Gadadhar & Dr. Carsten Janke, Curie Institute, Paris
|Product Type||Monoclonal Antibody|
|Source/Host||Purified from concentrated hybridoma tissue culture supernatant.|
|Immunogen/Antigen||Tubulin of the Ciliate Euplotes eluted from the 55kDa band of SDS gel.|
Western Blot: 1:2000
Detects K40 acetylation of α-tubulin; signal specifically increased by modification with tubulin acetyl transferase α-TAT1.
|Purity Detail||Protein G-affinity purified.|
|Formulation||Liquid. In PBS containing 10% glycerol and 0.02% sodium azide.|
|Isotype Negative Control|
|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. Tubulin acetylation was discovered on K40 of flagellar α-tubulin in Chlamydomonas reinhardtii and is generally enriched on stable microtubules in cells. It is located on the microtubule lumenal surface. As a result of its localization at the inner face of microtubules, K40 acetylation might rather affect the binding of microtubule inner proteins, a poorly characterized family of proteins. Functional experiments in cells have further suggested that K40 acetylation regulates intracellular transport by regulating the traffic of kinesin motors probably by indirect mechanisms. Acetyltransferase α-Tat1 (or Mec-17) specifically acetylate α-tubulin K40. Acetylation of tubulin by α-Tat1 accumulates selectively in stable, long-lived microtubules thus explaining the link between this posttranslational modication and stable microtubules in cells. However, the direct cellular function of K40 acetylation on microtubules is still unclear.
- Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium: A.M. Callen, et al.; Biol. Cell 81, 95 (1994)
- Where and when is microtubule diversity generated in Paramecium? Immunological properties of microtubular networks in the interphase and dividing cells: A. Fleury, et al.; Protoplasma 189, 37 (1995)
- Structural inheritance in Paramecium: ultrastructural evidence for basal body and associated rootlets polarity transmission through binary fission: F. Iftode & A. Fleury-Aubusson; Biol. Cell 95, 39 (2003)
- Investigating tubulin posttranslational modifications with specific antibodies: M.M. Magiera & C. Janke; In Methods Cell Biol. (Burlington: Academic Press) 115, 247 (2013)