PsbA | D1 protein of PSII, phosphorylated
AS13 2669 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] for higher plants
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KLH-conjugated synthetic peptide derived from available plant PsbA sequences with phosphorylated (T), including Arabidopsis thaliana UniProt: P83755, TAIR:AtCg00020, Oryza sativa P0C434 and other higher plant PsbA sequences
38 | 28-30 kDa
Dictos, Conifers, Monocts
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5 µg of total protein from 10 days old seedlings of Hordeum vulgare exposed to high light of 450 µmol to induce phosphorylation (1), or low light of 2 µmol (2), darkness (3) for three hours were extracted with Protein Extraction Buffer PEB (AS08 300). All three samples were treated with lambda protein phosphatase (NEB) to dephosphorylate the PsbA protein (Phosphatase-treated). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heated 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 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 anti-PsbA (AS05 084, first 3 and last 3 lanes on a blot) and anti-phosphorylated PsbA (AS13 2669, middle lanes) 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 (Agrisera 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 extreme femtogram range chemiluminescnce 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 minutes.
Antibodies are purified on a non-phosphorylated peptide.
The psbA gene has been cloned from many species of plants, green algae, and cyanobacteria. The psbA gene is located in the chloroplast genome and encodes for the D1 protein, a core component of Photosystem II. PsbA/D1 is rapidly cycled under illumination in all oxygenic photobionts. Tracking PsbA pools using the Global PsbA antibody can show the functional content of Photosystem II in a wide range of samples.
Alternative names: 32 kDa thylakoid membrane protein, photosystem II protein D1.
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