H+ATPase | Plasma membrane H+ATPase (chicken antibody)
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|Recommended dilution||1 : 1000-1 : 5000 (WB)|
|Expected | apparent MW||
95 kDa (Arabidopsis thaliana)
|Confirmed reactivity||Arabidopsis thaliana, Spinacia oleracea, Zea mays|
|Predicted reactivity||Angomonas deanei, Avena sativa, Brassica napus, Citrus limon, Coffea canephora, Cucumis sativus, Cucurbita moschata, Dunaliella spp, Eichhornia crassipes, Emiliana huxleyi, Glycine max (weak), Hordeum vulgare, Lactobacillus johnsonii, Laishamania braziliensis, Nicotiana tabacum, Oryza sativa, Solanum lycopersicon, Solanum tuberosum, Medicago truncatula, Mesembruanthemum crystallinum), Nepenthes alata, Nicotiana tabaccum, Nitrospira bacterium, Oryza sativa, Ostreococcus spp., Phaseolus acutifolius, Physocomitrella patens, Picea abies, Pinus thunbergii, Populus tremula, Pteris vittata, Ricinus communis, Saccharomyces cerevisiae, Solanum lycopersicum, Strigomonas culicis, Toxoplasma gondii, Triticum urartu, Trypanosoma cruzi, Zosteria marina, Vicia faba, Vigna angularis|
|Not reactive in||
VERY IMPORTANT: please, do not heat up your samples over 70°C as this might cause H+ATPase to precipitate and there will be no signal on your western blot.
to be added when available, antibody released in November 2014
10 µg of total protein from whole leaf extracts of Arabidopsis thaliana (1), Zea mays (2), Spinacia oleracea (3), extracted with Protein Extration Buffer, PEB (AS08 300), were boiled for 10 min. in 70°C and separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Blots were blocked immediately following transfer in 2% ECL blocking reagent (GE Healthcare) 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: 2 500 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in 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 for 1h at room temperature with agitation. The blots were washed as above and developed for 5 minutes according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
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