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anti-Tubulin (glycylated), pAb (Gly-pep1)

AG-25B-0034-C100 100 µg INQ
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Properties
Source/Host Rabbit
Immunogen/Antigen Synthetic peptide corresponding to D431EQGEFE(G-COOH*)EEEG441-NH2 of human Tubulin β-2A chain (*secondary glycine branched from γ-carboxyl group of glutamate as isopeptide bond).
Application Western Blot: (1:10'000)
Immunocytochemistry: (1:5'000)
Immunoprecipitation: (1:200)
Crossreactivity Dog, Human, Mouse
Specificity This antibody recognizes mono or bi-glycylated Tubulins. The activity of glycylating enzymes (TTLL3 and TTLL8) in cultured cells leads mainly to the modification of α- and β-tubulin, but also of other, yet unidentified protein substrates also detected by the antibody Gly-pep1. In immunofluorescence labeling, the antibody strongly labels glycylated microtubules (Gadadhar, et al.; 2017).
Purity ≥95% (SDS-PAGE)
Purity Detail Epitope-affinity purified.
Concentration 1mg/ml
Formulation Liquid. In PBS containing 0.02% sodium azide.
Accession Number Q13885
Product Type Polyclonal Antibody
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.
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Product Description

Microtubules are key cytoskeletal elements that are found in all eukaryotic cells. Microtubules fulfil a large range of different functions, which are thought to be controlled by the ‘tubulin code’ – mechanism to generate distinct microtubule identities. One mechanism to label specific microtubules are tubulin posttranslational modifications (PTMs), of which a large variety exists. One of these modifications is glycylation, which is generated by the addition of secondary (branched) glycine chains to the main (primary) peptide chain of the protein. The length of these branch chains can vary from one to more than 20 glycine residues. Glycylation is catalysed by the enzymes TTLL3, TTLL8 and TTLL10 from the tubulin tyrosine ligase-like (TTLL) family. Especially TTLL3 and TTLL8 are essential for the initiation of the glycylation because the generate the nascent glycine chain.

The Gly-pep1 antibody was raised against a peptide mimicking beta2-tubulin (TUBB2A) with a single glycine branch on E437. The antibody specifically detects glycylated tubulin, and also other yet unknown glycylation substrates in cells as well as in tissues. As glycyation of microtubules is particularly found in cilia and flagella, Gly-pep1 labels motile cilia as well as primary cilia (Gadadhar et al., 2017)


Product References

Tubulin glycylation controls primary cilia length: S. Gadadhar, et al.; J. Cell Biol. (Article in press) (2017)
Immunoblot analysis of protein glycylation using anti-Tubulin (glycylated), pAb (Gly-pep1) (Prod. No. AG-25B-0034). <b>Method:</b> HEK-293T cells are grown in standard culture conditions, transfected with plasmids expressing the glycylases TTLL3 and TTLL8 and are run on a 10% SDS-PAGE. Lysate of mouse testes was used as a positive control for glycylated tubulin. The proteins are transferred to a nitrocellulose membrane and detected by standard immunoblot protocol using anti-Tubulin (glycylated), pAb (Gly-pep1) (1:20,000) in TBS containing 0.1% Tween-20 for washing steps and 2.5% fat free milk for antibody incubation. In the untransfected HEK-293T cell extracts, no tubulin is detected, even on high exposure. After expression of TTLL3 glycylase, predominantly &beta;-tubulin is detected with Gly-pep1, while expression of TTLL8 generates Gly-pep1-positive &alpha;-tubulin and other, yet unidentified substrates. Lysate of testes also contains a heterogeneous mixture of different glycylated proteins, inlcuding &alpha; and &beta;-tubulin. <i>Picture courtesy of Sudarshan Gadadhar and Carsten Janke, Institut Curie, Paris.</i>
Immunoblot analysis of protein glycylation using anti-Tubulin (glycylated), pAb (Gly-pep1) (Prod. No. AG-25B-0034). Method: HEK-293T cells are grown in standard culture conditions, transfected with plasmids expressing the glycylases TTLL3 and TTLL8 and are run on a 10% SDS-PAGE. Lysate of mouse testes was used as a positive control for glycylated tubulin. The proteins are transferred to a nitrocellulose membrane and detected by standard immunoblot protocol using anti-Tubulin (glycylated), pAb (Gly-pep1) (1:20,000) in TBS containing 0.1% Tween-20 for washing steps and 2.5% fat free milk for antibody incubation. In the untransfected HEK-293T cell extracts, no tubulin is detected, even on high exposure. After expression of TTLL3 glycylase, predominantly β-tubulin is detected with Gly-pep1, while expression of TTLL8 generates Gly-pep1-positive α-tubulin and other, yet unidentified substrates. Lysate of testes also contains a heterogeneous mixture of different glycylated proteins, inlcuding α and β-tubulin. Picture courtesy of Sudarshan Gadadhar and Carsten Janke, Institut Curie, Paris.
 Immunofluorescence staining using anti-Tubulin (glycylated), pAb (Gly-pep1) (Prod. No. AG-25B-0034) of multiciliated ependymal cells and cells with primary cilia. <b>Method:</b> Radial glial cells isolated from newborn wildtype mice, and the MDCK cell line were serum starved to induce ciliogenesis. Cells were fixed with a microtubule-stabilizing protocol and stained with anti-Tubulin (glycylated), pAb (Gly-pep1) (1:5,000; red), anti-&alpha;-Tubulin (acetylated), mAb (TEU318) (AG-20B-0068) (green) and DAPI (blue). Gly-pep1 staining is observed specifically on the cilia. <i>Picture courtesy of Sudarshan Gadadhar and Carsten Janke, Institut Curie, Paris.</i>
Immunofluorescence staining using anti-Tubulin (glycylated), pAb (Gly-pep1) (Prod. No. AG-25B-0034) of multiciliated ependymal cells and cells with primary cilia. Method: Radial glial cells isolated from newborn wildtype mice, and the MDCK cell line were serum starved to induce ciliogenesis. Cells were fixed with a microtubule-stabilizing protocol and stained with anti-Tubulin (glycylated), pAb (Gly-pep1) (1:5,000; red), anti-α-Tubulin (acetylated), mAb (TEU318) (AG-20B-0068) (green) and DAPI (blue). Gly-pep1 staining is observed specifically on the cilia. Picture courtesy of Sudarshan Gadadhar and Carsten Janke, Institut Curie, Paris.
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