UGPase | UDP-glucose pyrophosphorylase (cytoplasm marker) (Hordeum vulgare)
AS14 2813 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, H.vulagre, Z.mays
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His-tagged, full length Hordeum vulgare UGPase, overexpressed and purified from E.coli, UniProt: Q43772.1
Bambusa oldhamii, Brassica pekinensis, Brassica rapa, Capsicum annuum, Cucumis sativus, Dendrobium catenatum, Dendrocalamus sinicus, Glycine max, Gossipium hirsutum, Lycopersicum esculentum, Lycopersicum chilense, Marchantia polymorpha, Oryza sativa, Picea glauca, Populus sp., Solanum tuberosum, Populus tremula, Ricinus communis, Saccharum officinarum, Vitis vinifera, for more species, please inquire
10 µg of total protein from Arabidopsis thaliana leaf (1) , Hordeum vulgare leaf (2), Zea mays leaf (3), recombinant UGPase 0.5 pmol (4), were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 10 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602, Agrisera) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 10 seconds.
UDP-glucose pyrophosphorylase (UGPase, UDPGP) E.C=22.214.171.124. is a key enzyme of synthesis of sucrose, cellulose and other saccharides. There are two cytoplasmic isoforms of UGPase-A (which share 94 % identity on amino acid level) and one chloroplastic UGPase-B isoform in Arabidopsis thaliana which share ca. 10-11 % of identity (Kleczkowski et al. 2011).
Alternative name: UTP--glucose-1-phosphate uridylyltransferase.
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