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AdipoGen Life Sciences
anti-β-Tubulin (β-monoE), pAb (IN115)
460
CHF
CHF 460.00
In stock
AG-25B-0039-C05050 µgCHF 460.00
Figure 1: Immunoblot analysis of protein glutamylation with anti-β-Tubulin (β-monoE), pAb (IN115) (Prod. No. AG-25B-0039). Method: HEK-293T cells are grown in standard culture conditions, transfected with plasmids expressing the different
glutamylases and are run on a 10% SDS-PAGE (specific protocol to separate α- and β-tubulin (Magiera & Janke, 2013)). Tubulin purified from pork brain was used as a positive control for glutamylated tubulin. The proteins are transferred to a nitrocellulose membrane and detected by standard immunoblot protocol using the β-monoE (1:5,000) in PBS containing 0.1% Tween-20 for washing steps and 2.5% fat free milk for antibody incubation. In untransfected HEK-293T cell extracts, no tubulin is detected, even on high exposure. After expression of TTLL4, predominantly β-tubulin is detected with β-monoE, but the antibody also faintly detects the heterogeneous mixture of different, yet unidentified substrates modified by TTLL4. In cells expressing TTLL5 and TTLL6, the β-monoE detects only β-tubulin, while expression of TTLL7 generates β-monoE-positive α- and β-tubulin. In the purified brain tubulin, the antibody specifically detects glutamylation of β-tubulin. Picture courtesy of Sudarshan Gadadhar, Shreyangi Chakraborty, Mariya Genova and Carsten Janke, Institut Curie, Orsay.
Figure 2: Immunoblot analysis of glutamylation by brain-specific TTLLs with anti-β Tubulin (β-monoE), pAb (IN115) (Prod. No. AG-25B-0039). Method: Lysates of brain from wildtype, Ttll1-KO, Ttll7-KO and Ttll1-Ttll7-double KO mice are run on a 10% SDS-PAGE and subjected to an immune blot analysis with β-monoE antibody as detailed in Figure 1. The antibody specifically detects glutamylation of β-tubulin in the wildtype and Ttll1-KO brain lysates as TTLL1 modifies only α-tubulin. In the lysates of Ttll7-KO and Ttll1-Ttll7-double KO mouse brains, the antibody does not detect glutamylation of β-tubulin as TTLL7 is specific to β-tubulin, thus establishing the specificity of the antibody. Equal loading of the samples was confirmed with clone 12G10, an α-tubulin-specific antibody. Picture courtesy of Sudarshan Gadadhar, Shreyangi Chakraborty, Mariya Genova and Carsten Janke, Institut Curie, Orsay.
| Product Details | |
|---|---|
| Product Type | Polyclonal Antibody |
| Properties | |
| Source/Host | Rabbit |
| Immunogen/Antigen | Synthetic peptide corresponding to D431EQGEFE(E-COOH*)EEEG441-NH2 of human tubulin β-2A chain (*secondary glutamate branched from γ-carboxyl group of glutamate as isopeptide bond). |
| Application |
Western Blot: (1:2000) Immunocytochemistry: (1:5000) Immunohistochemistry Immunoprecipitation Optimal conditions must be determined individually for each application. |
| Crossreactivity |
Human Mouse |
| Specificity |
Recognizes the posttranslational modification glutamylation with specific reactivity for the GE(*-E)F motive. Because the sequence GEF is specific to several β-tubulin isotypes, the antibody selectively detects mono-glutamylated β-tubulin. |
| Purity | ≥95% (SDS-PAGE) |
| Purity Detail | Epitope-affinity purified. |
| Concentration | 1mg/ml |
| Formulation | Liquid. In PBS containing 0.02% Proclin 300. |
| Isotype Negative Control | |
| Other Product Data |
Recognizes predominantly mono-glutamylated β-tubulin. However, in cells overexpressing enzymes that can modify both α- and β-tubulin, it might detect glutamylation on α-tubulin as well. Requires GE(*-E)F motive, which is present in several β-tubulin isotypes. No other specificity to particular tubulin isotypes nor to tubulin from particular species are observed. The use of the antibody at too high concentrations obscures its specificity in immunofluorescence. |
| Accession Number | Q13885 |
| Shipping and Handling | |
| Shipping | BLUE ICE |
| Short Term Storage | +4°C |
| Long Term Storage | -20°C |
| Handling Advice |
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. |
| Documents | |
| MSDS |
 Download PDF |
| Product Specification Sheet | |
| Datasheet |
Download PDF |
Description
Polyglutamylation is an evolutionarily conserved protein post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. The most prominent substrate is tubulin, the building block of microtubules (MTs). Polyglutamylation has been found on both, α- and β-tubulin. It is catalyzed by multiple enzymes that belong to the family of tubulin tyrosine-ligase like (TTLL) enzymes. These TTLLs have substrate (α- vs β-tubulin) as well as reaction (initiation vs elongation) specificities. These subtle changes in levels of polyglutamylation on specific tubulin isoforms could in turn be influencing the diverse MT functions that are regulated by polyglutamylation. The β-monoE antibody selectively labels glutamylation of β-tubulin due to its specificity to a sequence motif (GE(*-E)F) that only exists in β-tubulin isotypes. It allows to specifically detect β-tubulin glutamylation in brain tubulin, where other antibodies predominantly detect α-tubulin glutamylation.
Product References
- Distinct roles of alpha- and beta-tubulin polyglutamylation in controlling axonal transport and in neurodegeneration: S. Bodakuntla, et al.; EMBO J. 40, e108498 (2021)
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