ARF1 | ADP-ribosylation factor 1
AS08 325 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, C.reinhardtii, Elaeis sp. , L. longiflorum, M. truncatula, N. tabacum, O. sativa, P. hybrida cv Mitchell, P. patens, S. tuberosum | Cellular [compartment marker] of Golgi in immunolocalization and COP1 in Western blot
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21 kDa (Arabidopsis thaliana)
Brassica juncea, Brassica napus, Capsella rubella, Capsicum annum, Cucumis sp., Daucus carota, Elaeis guineensis, Glycine max, Helleborus orientalis, Hordeum vulgare, Medicago truncatula, Nicotiana benthamina, Populus trichocarpa, Zea mays
50 µg of total protein from (1) Nicotiana tabacum protoplast total protein, (2) Arabidopsis thaliana protoplast soluble protein, (3) Arabidopsis thaliana protoplast total protein were separated on 10 % SDS-PAGE and blotted 2h to nitrocellulose (Semi-dry, 200mA). Filters were blocked over night with 5% low-fat milk powder in TBS and probed with anti-Sec21p antibodies (AS08 327, 1:1000, 1h) and secondary anti-rabbit (1:20000, 1 h) antibody (HRP) in TBS-Tween (recommended secondary antibody AS09 602). Signal was detected with standard ECL andexposure time for this image was 1 minute.
Protoplasts were extracted in 50mM Tris, 10 mM EDTA and Triton X100, 0.02%.
Specificity testing of rabbit anti-ARF1 serum. Immunofluorescence labelling of rabbit anti-ARF1 antibody (red) in 5-day-old root epidermal cells of the Arabidopsis thaliana ecotype Columbia-0 (WT) or seedlings expressing the ADP-RIBOSYLATION FACTOR 1 (AtARFA1c; accession At2g47170) fused to EGFP (green) (Xu, J. and Scheres, B. 2005. Plant Cell 17, 525-536). The rabbit anti-ARF1 antibody was diluted 1:1000 and the secondary antibody, donkey anti-rabbit CY5-coupled (Jackson ImmunoResearch) was diluted 1:300. The nuclei were stained with DAPI (blue). Note the co-labelling of ARF1-GFP with the anti-ARF1 antibody (arrowheads) and the additional labelling (potentially of other ARF1 variants) by the anti-ARF1 antibody (arrows). The antibody staining permeability was limited to the 1-2 outermost layers of the whole-mounted root tips.
Courtesy of Dr. Anna Gustavsson and Dr. Markus Grebe
Cellular [compartment marker] of Golgi in immunolocalization and COP1 in western blot
References describing immunolocalization (IF) and (IG) studies:
The ARF1 protein is localized to the Golgi apparatus and has a central role in intra-Golgi transport. It is a small GTPase that undergoes a GDP/GTP nucleotide exchange cycle and it is an important regulator of cellular trafficking.
Lynch et al. (2017). Multifaceted plant responses to circumvent Phe hyperaccumulation by downregulation of flux through the shikimate pathway and by vacuolar Phe sequestration. Plant J. 2017 Dec;92(5):939-950. doi: 10.1111/tpj.13730.
Vincent et al. (2017). A genome-scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association. Plant J. 2017 Dec;92(6):1132-1142. doi: 10.1111/tpj.13749.
Ma et al. (2016). Phosphatidylserine Synthase Controls Cell Elongation Especially in the Uppermost Internode in Rice by Regulation of Exocytosis. PLoS One. 2016 Apr 7;11(4):e0153119. doi: 10.1371/journal.pone.0153119. eCollection 2016.
Yüzbaşıoğlu et al. (2016). Functional specialization of Arf paralogs in nodules of model legume, Medicago truncatula. Plant Growth Regul. DOI: 10.1007/s10725-016-0227-2.
Marais et al. (2015). The Qb-SNARE Memb11 interacts specifically with Arf1 in the Golgi apparatus of Arabidopsis thaliana. J Exp Bot. 2015 Jul 24. pii: erv373.
Wang et al. (2015). UDP-D-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the trans-Golgi network/early endosome in Arabidopsis thaliana root epidermal cells. J. Plant Res. 2015 May 27. (immunogold application)