H+ATPase | Plasma membrane H+ATPase (chicken antibody)
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KLH-conjugated synthetic peptide derived from available di and monocot, fern, mosses and algal plasma membrane ATPase sequences including Arabidopsis thaliana ATPase 1 (At2g18960) and ATPase 2,3,4,6,7,8,9 of Arabidopsis thaliana and hydrogen ATPase of Chlamydomonas reinhardtii (Q9FNS3)
95 kDa (Arabidopsis thaliana)
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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 AS10 1489) 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).
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.
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.