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anti-Polyglutamylation Modification, mAb (GT335)

AG-20B-0020-C100 100 µg INQ
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Product Data
Synonyms Polyglutamylated Tubulin; Glutamylated Tubulin; Postranslational Protein Glutamylation
Properties
Clone GT335
Isotype Mouse IgG1κ
Source/Host Purified from concentrated hybridoma tissue culture supernatant.
Immunogen/Antigen Octapeptide EGEGE*EEG, modified by the addition of two glutamyl units onto the fifth E (indicated by an asterisk).
Application Electron Microscopy (see Lit. 3)
Immunocytochemistry: (1:2000)
Immunoprecipitation
Western Blot: (1:4000)
Optimal conditions must be determined individually for each application.
Crossreactivity All
Specificity Recognizes the posttranslational modification (poly)glutamylation on proteins. Reacts with polyglutamylated α- and β-tubulin.
Purity ≥95% (SDS-PAGE)
Purity Detail Protein G-affinity purified.
Concentration 1mg/ml
Formulation Liquid. In PBS containing 10% glycerol and 0.02% sodium azide.
Other Product Data Recognizes most forms of polyglutamylated tubulin (α- and β-tubulin), independent of the length of the glutamate side chains. No specificity to particular tubulin isoforms nor to tubulin from particular species are observed. Detects also other (poly)glutamylated proteins. Since no consensus modification site is known for protein (poly)glutamylation, the detection is not sequence-specific. However, an acidic environment of the modification site is required.
The use of the antibody at too high concentrations obscures its specificity in immunofluorescence.
Product Type Monoclonal 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.
Documents
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Product Description

Polyglutamylation is a post-translational modification in which glutamate side chains of variable lengths are formed on the modified protein. It is evolutionarily conserved and the most prominent substrate is tubulin, the microtubule (MT) building block. Polyglutamylation has been proposed to be involved in the functional adaptation of MTs, as it occurs within the carboxy-terminal tubulin tails that participate directly in the binding of many structural and motor MT-associated proteins. The recent identification of new substrates of polyglutamylation indicates that this post-translational modification could be a potential regulator of diverse cellular processes and be involved in cell cycle and cell proliferation.

Product References

  1. Distribution of glutamylated alpha and beta-tubulin in mouse tissues using a specific monoclonal antibody, GT335: A. Wolff, et al.; Eur. J. Cell Biol. 59, 425 (1992)
  2. Polyglutamylation of nucleosome assembly proteins: C. Regnard, et al.; J. Biol. Chem. 275, 15969 (2000)
  3. Glutamylated tubulin: diversity of expression and distribution of isoforms: M.L. Kann, et al.; Cell Motil. Cytoskeleton 55, 14 (2003)
  4. Polyglutamylation Is a Post-translational Modification with a Broad Range of Substrates: J. van Dijk, et al.; J. Biol. Chem. 283, 3915 (2008)
  5. Unique post-translational modifications in specialized microtubule architecture: K. Ikegami & M. Setou; Cell Struct. Funct. 35, 15 (2010) (Review)
  6. Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells: S. Zink , et al.; J. Cell Sci. 125, 5998 (2012)
  7. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly: T. Kobayashi, et al.; J. Cell Biol. 204, 215 (2014)
  8. CNS axons globally increase axonal transport after peripheral conditioning: F.M. Mar, et al.; J. Neurosci. 34, 5965 (2014)
  9. Microtubule detyrosination guides chromosomes during mitosis: M. Barisic, et al.; Science 348, 6236 (2015) (Supplement)
  10. SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture: M. Hilbert, et al.; Nat. Cell Biol. 18, 393 (2016)
  11. Loss of RPGR glutamylation underlies the pathogenic mechanism of retinal dystrophy caused by TTLL5 mutations: X. Sun, et al.; PNAS 113, E2925 (2016)
Immunofluorescence analysis of tubulin glutamylation with anti-Polyglutamylation Modification, mAb (GT335) (Prod. No. AG-20B-0020).<br />
HeLa cells grown in standard culture conditions are transfected with GFP-fusion expression plasmids for the polyglutamylases TTLL4 and TTLL6. Glutamylated microtubules are detected with anti-Polyglutamylation Modification, mAb (GT335). Only in the cells expressing glutamylases (red arrowheads), strong microtubule labelling is detected, whereas in non-transfected cells (white arrowheads), only very weak labelling is seen. Note that the level of endogenous microtubule glutamylation can vary between different cell lines.
<i>Picture courtesy of Dr. Carsten Janke, Curie Institute, Paris.</i>
Immunofluorescence analysis of tubulin glutamylation with anti-Polyglutamylation Modification, mAb (GT335) (Prod. No. AG-20B-0020).
HeLa cells grown in standard culture conditions are transfected with GFP-fusion expression plasmids for the polyglutamylases TTLL4 and TTLL6. Glutamylated microtubules are detected with anti-Polyglutamylation Modification, mAb (GT335). Only in the cells expressing glutamylases (red arrowheads), strong microtubule labelling is detected, whereas in non-transfected cells (white arrowheads), only very weak labelling is seen. Note that the level of endogenous microtubule glutamylation can vary between different cell lines. Picture courtesy of Dr. Carsten Janke, Curie Institute, Paris.
Western blot analysis of protein glutamylation with MAb to polyglutamylation modification (GT335) (Prod. No. AG-20B-0020).<br />
<b>Method:</b> HeLa cells grown in standard culture conditions are lysed in SDS sample buffer and run on 10% SDS-PAGE. The proteins are transferred to nitrocellulose membrane and detected by standard Western blot protocol, using the MAb to polyglutamylation modification (GT335) (1:1’000) in TBS containing 0.1% Tween 20 for washing steps and 5% fat free milk for antibody incubation.<br />
In HeLa cells, only two non-tubulin proteins are detected (Regnard et al., 2000; van Dijk et al., 2008), whereas after
the expression of either TTLL4 or TTLL6 polyglutamylases (van Dijk et al., 2007), tubulin and additional proteins are
detected. Brain tubulin is highly polyglutamylated and therefore strongly detected with the MAb to polyglutamylation modification (GT335).<br />
<i>Picture courtesy of Dr Carsten Janke, Curie Institute, Paris</i>
Western blot analysis of protein glutamylation with MAb to polyglutamylation modification (GT335) (Prod. No. AG-20B-0020).
Method: HeLa cells grown in standard culture conditions are lysed in SDS sample buffer and run on 10% SDS-PAGE. The proteins are transferred to nitrocellulose membrane and detected by standard Western blot protocol, using the MAb to polyglutamylation modification (GT335) (1:1’000) in TBS containing 0.1% Tween 20 for washing steps and 5% fat free milk for antibody incubation.
In HeLa cells, only two non-tubulin proteins are detected (Regnard et al., 2000; van Dijk et al., 2008), whereas after the expression of either TTLL4 or TTLL6 polyglutamylases (van Dijk et al., 2007), tubulin and additional proteins are detected. Brain tubulin is highly polyglutamylated and therefore strongly detected with the MAb to polyglutamylation modification (GT335).
Picture courtesy of Dr Carsten Janke, Curie Institute, Paris
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