SBPase | Sedoheptulose-1,7-bis phosphatase
AS15 2873 | clonality: polyclonal | host: rabbit | reactivity: A.thaliana, Chlamydomonas sp. (strain W80), H.vulgare, Z. mays
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|Recommended dilution||1 : 1000-1 : 5000 (WB)|
|Expected | apparent MW||
|Confirmed reactivity||Arabidopsis thaliana, Chlamydomonas sp. (strain W80), Hordeum vulgare, Zea mays|
|Predicted reactivity||Arabidopsis lyrata, Auxenochlorella protothecoides, Brachypodium distachyon , Brassica rapa, Coccomyxa subellipsoidea C-169 GN, Cucumis sativus, Dunaliella tertiolecta, Ectocarpus siliculosus, Genlisea aurea, Glycine soja, Gossypium raimondii , Marchantia polymorpha, Medicago truncatula, Mesostigma viride, Morus notabilis, Nicotiana tabacum, Oryza sativa, Physcomitrella patens, Populus trichocarpa, Ricinus communis, Sorghum bicolor, Spinacia oleracea, Tetraselmis sp. GSL018, Theobroma cacao, Triticum aestivum, Triticum urartu, Zea mays, Volvox carteri|
|Not reactive in||Cyanobacteria|
to be added when available antibody available in November 2015
10 µg of total protein from Arabidopsis thaliana leaf (1) , SBPase protein standard AS15 2873S (2) 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 TMA-6 (Lumigen) 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 30 seconds.
5 or 10 µg of total protein from Chlamydomonas reinhardtii extracted with 10 mM Tris/HCl pH7.5, 80 mM NaCl, 1 mM EDTA, 1 % (w/v) Glycerol, 1 mM DTT, 1x protease inhibitor cocktail (Roche) and denatured in SDS sample buffer for 5 min. at 95°C were separated on 12 % SDS-PAGE and blotted over night to nitrocellulose using tank transfer. Blots were blocked with 3 % milk; for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 500 in blocking buffer for 1h at RT with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 5 min. 5x in TBS-T (0.05 %) at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated from Agrisera AS09 602) diluted to 1:25 000; for 1h at RT with agitation. The blot was washed as above and developed using Luminol-H2O2 and p-Coumaric acid. Exposure time of X-ray film was 1 hour.
Courtesy of Dr. Sebastian Mahlow, Prof. Maria Mittag, Institute of General Botany and Plant Physiology, University of Jena, Germany
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