ADGP | ADP-glucose pyrophosphorylase
AS11 1739 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, C. reinhardtii, H. vulgare, N. tabacum, Polytomella sp., T. aestivum, Z. mays
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49.4 | 52 kDa
10 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3) total protein from all the samples 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 keep on ice before loading. Protein samples were separated on NuPAGE 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted for 1h to 1.5h on 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: 5 000 (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) 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 TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
2D gel electrophoresis was performed to exclude cross-reactivity of this antibody to Rubisco
Courtesy of Lauri Nikkanen, University of Turku, Finland
western blot on algal samples
Total proteins (40 µg) from Chlamydomonas reinhardtii strain CC-124 (1), Chlamydomonas reinhardtii strain CC43-48 (STA6) (2) and Polytomella sp. Pringsheim 198.80 (3) were separated on a 5-12% acrylamide/6M urea SDS-PAGE and blotted onto nitrocellulose membrane. Blots were blocked in T-TBS (0.1% Tween in Tris Buffer Saline) containing 5% low fat milk, 1 h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 500 for 1h at RT with agitation. The antibody solution was decanted and the blot was washed twice for 15 min in T-TBS, RT with agitation. Blot was incubated in secondary antibody diluted to 1: 25 000 (goat anti-rabbit IgG, horse radish peroxidase conjugated, Agrisera, AS09 602), 1h at RT with agitation. The blot was washed as above and developed using the homemade enhanced chemiluminescence system (Durrant, Nature 346: 297, 1990). Images of the blots were obtained using a CCD imager (ImageQuant LAS4000).
Courtesy of Ariane Atteia, CNRS/Aix-Marseille Université, France
Cross reactivity of this antibody to Rubisco has been excluded using 2D gel electrophoresis.
Antigen used to elicit this antbody has very poor conservation in large subunit of ADP-glucose pyrophosphorylase.
ADP-glucose pyrophosphorylase (AGPase) catalyses the first committed step in the synthesis of transient starch in chloroplasts and storage starch in amyloplasts in plants (Tetlow et al., 2004). AGPase in higher plants is composed of two distinct subunits encoded by separate genes, forming an L2S2 heterotetramer. The large subunits (L) are modulators of allosteric activity, while the small subunits (S) are the catalytic subunits (Kim et al., 2007). Synonymes:ADP-glucose pyrophosphorylase, ADP-glucose synthase, AGPase B, Alpha-D-glucose-1-phosphate adenyl transferase
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