AS07 267 | Clonality: Polyclonal | Host: Rabbit | Reactivity: higher plants and algae
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|Recommended dilution||1 µg/ml (ELISA), 2 µg/10 ml incubation buffer (WB)|
|Expected | apparent MW||
10-100 for various glycoproteins
|Confirmed reactivity||Higher plants and algae|
|Predicted reactivity||Higher plants|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
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
|Selected references||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.
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
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