AS07 267 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Higher plants and algae
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xylose residues bound to the N-glycan in beta 1,2
10-100 for various glycoproteins
Total cell extract from Arabidopsis thaliana wild type (1) and cell extracts from different mutants defective in the complex N-glycan maturation pathway (2-5) (data not published yet).
Primary antibody has been used at 2 µg/10 ml of incubation buffer. Detection has been done using ECL.
Dot blot reaction of anti-Fucose and anti-Xylose antibodies with various controls: Avidin (Fuc+/Xyl+), Fetuin (Fuc-/Xyl-), PLA2 (Fuc+/Xyl-) and Mur1-2 (Fuc-/Xyl+). 2 µl of each extract were spotted on a nitrocellulose membrane, placed on top of 2 WHATMAN filters (one soaked in TBS-T) and dried for 1.5 h at RT. The mem-brane was blocked for 30 min with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and incubated with anti-Fucose(1) (AS07 268, 1:1000) or anti-Xylose(2) (AS07 267, 1:1000) for 30 min and then with secondary anti-rabbit(1:1000) antibody (ALP conjugated, recommended secondary antibody AS09 607). Membrane was washed with TBS-T 3 x 10 minutes before reaction development using alkaline phosphatase reagent BCIP®/NTB premixed solution (Sigma, Prod. No. B6404).
Please follow this link for a more detailed Dot-Blot protocol
Beta (1,2) xylose is present exclusively in plant N-glycans so antibodies against this sugar moiety should not cross-react with any mammal glycoprotein.
This antibody do not bind free D-xylose. This antibody does not seem to work in immunolocalization.
Negative control: Fetuin, a glycoprotein containing fucose linked in alpha 1.6 and no xylose, Sigma, product number F3385.
Positive control: Type II - horseradish peroxidase which contains β1.2 Xylose and α1.3 fucose, Sigma, product number P8250
This antibody specifically cross-reacts against xylose residues bound to the protein N-glycans in beta1,2. This residue is characterisitc of the plant protein N-glycans and is absent in protein N-glycans from animals. This residue is added in the Golgi apparatus.
Jansing et al. (2018). CRISPR/Cas9-mediated knockout of six glycosyltransferase genes in Nicotiana benthamiana for the production of recombinant proteins lacking β-1,2-xylose and core α-1,3-fucose. Plant Biotechnol J. 2018 Jul 3. doi: 10.1111/pbi.12981.
Nakanishi et al. (2017). Protection of Human Colon Cells from Shiga Toxin by Plant-based Recombinant Secretory IgA. Sci Rep. 2017 Apr 3;7:45843. doi: 10.1038/srep45843. (ELISA)
Hanania et al. (2017). Establishment of a tobacco BY2 cell line devoid of plant specific xylose and fucose as a platform for the production of biotherapeutic proteins. Plant Biotechnol J. 2017 Feb 3. doi: 10.1111/pbi.12702.
Ebert et al. (2015). Identification and Characterization of a Golgi-Localized UDP-Xylose Transporter Family from Arabidopsis. Plant Cell. 2015 Mar 24. pii: tpc.114.133827.
Mathieu-Rivet et al. (2013). Exploring the N-glycosylation pathway in Chlamydomonas reinhardtii unravels novel complex structures. Mol Cell Proteomics, Aug 2.