PsbA | D1 protein of PSII, phosphorylated
AS13 2669 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] for higher plants
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|Recommended dilution||1 : 10 000 (WB)|
|Expected | apparent MW||
38 | 28-30 kDa
|Confirmed reactivity||Arabidopsis thaliana, Hordeum vulgare, Zea mays|
Dictos, Conifers, Chlamydomonas reinhardtii, Monocts
|Not reactive in||Cyanobacteria|
|Additional information||This antibody is detecting phosphorylated PsbA protein.|
|Selected references||Xing et al. (2017). Deletion of CGLD1 Impairs PSII and Increases Singlet Oxygen Tolerance of Green Alga Chlamydomonas reinhardtii. Front. Plant Sci., 15 December 2017.
Li et al. (2015). Effect of hydrogen sulfide on D1 protein in wheat under drought stress. Acta Physiologiae Plantarum November 2015, 37:225.
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 (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 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 TMA-6 (Lumigen) 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.
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