H+ATPase | Plasma membrane H+ATPase (100 ĩl)
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KLH-conjugated synthetic peptide exposed to cytoplasm in H+ATPase model, derived from available di and monocot, fern, mosses and algal plasma membrane ATPase sequences including Arabidopsis thaliana ATPase 1 (UniProt: P20649, TAIR:At2g18960) and ATPase 2 (UniProt: P19456 , TAIR:At4g30190),3 (UniProt: P20431, TAIR:At5g57350),4 (UniProt: Q9SU58, TAIR:At3g47950),6 (UniProt: Q9SH76, TAIR:At2g07560),7 (UniProt: Q9LY32, TAIR:At3g60330),8 (UniProt: Q9M2A0, TAIR:At3g42640),9 (UniProt: Q42556, TAIR:At1g80660), 11 (UniProt: Q9LV11, TAIR:At5g62670) of Arabidopsis thaliana and hydrogen ATPase of Chlamydomonas reinhardtii (Q9FNS3)
95 kDa (Arabidopsis thaliana)
Mesembruanthemum crystallinum, Solanum tuberosum; including Avena sativa, Hordeum vulgare, conifers (Pinus thunbergii), Mosses (Physocomitrella patens), Algae (Dunaliella spp., Ostreococcus spp.), Saccharomyces cerevisiae
20 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3), Nicotiana tabaccum plasma membrane fraction, 2.5 µg (4), 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 Advance 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: 5 000 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 1:20 000 in 2% ECL Advance blocking solution for 1h at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL Advance 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). Exposure time was 2 min.
Plasma membrane H+ATPase localization inArabidopsis thaliana roots.
Arabidopsis thaliana, elongation zone, H+ATPase (green). Arabidopsis thaliana roots were fixed in para-formaldehyde for 30 minutes. Tissue cleaning has been performed before immunolocalization. Anti-rabbit H+ATPase | plasma membrane primary antibody diluted in 1: 300 and anti-rabbit IgG secondary antibody conjugated with Alexa 555. Co-staining with DAPI visualized nucleus (blue color). Scale bar – 100 µm.
Courtesy Dr. Taras Pasternak, Freiburg University
This product can be sold containing ProClin if requested.
The Plasma Membrane H+ATPase is a family of proteins of ca. 100 kDa that are believed to be exclusive to the plasma membranes of plants and fungi. The protein is anchored within biological membrane which creates an electrochemical gradient used as an energy source and is essential for uptake of most metabolites and plant responses to environment, for example movement of leaves.
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