PsbA | D1 protein of PSII, C-terminal (affinity purified)
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KLH-conjugated synthetic peptide derived from available plant, algal and cyanobacterial PsbA sequences, including Arabidopsis thaliana UniProt: A4QJR4, TAIR: AtCg00020 , Oryza sativa P0C434, Populus alba Q14FH6, Physcomitrella patens Q6YXN7, Chlamydomonas reinhardtii P07753, Synechocystis sp. P14660 and many others
38 | 28-30 kDa
Algae (brown and red), Dictos, Conifers, Cyanobacteria, Medicago sativa, Pisum sativum, Triticum aestivum, cellular [compartment marker] of thylakoid membrane.
2 µg of total protein from samples such as Arabidopsis thaliana leaf (1) , Hordeum vulgare leaf (2), Chlamydomonas reinhardtii total cell (3), Synechococcus sp. PCC7942 total cell (4), were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70°C for 5 min and keept on ice before loading. Protein samples were separated on 4-12% Bolt Plus gels, LDS-PAGE and blotted for 70 minutes to 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 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 (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602, Agrisera) 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 ECL detection reagent according to the manufacturer's instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.
Due to biology of PsbA (D1) protein a number of degradation products can apprear in a sample and may be observed when using anti-PsbA antibodies, including products having apparent molecular weights of 24kDa and 16kDa. D1 degradation is a complex set of events and the products observed can be influenced by both the extraction procedure and the physiology of the cells prior to harvest. Third, cross-linking may occur between D1 and cytochrome b559, shifting the protein higher in the gel. In cyanobacteria (PCC7942), three different bands were competed out by preincubating the antibody with the PsbA free peptide, indicating that all bands are indeed PsbA and its precursors or breakdown products. Competition assays were also performed with spinach and Chlamydomonas, confirming the identity of PsbA bands.
Anti-PsbA antibodies will not detect D2 protein, as the peptide used to generate PsbA antibodies has no homology to the D2 sequence.
This antibody will detect the phosphorylated form of D1 as an alternate band to the main band on a high resolution gel.
The antibody will bind to cross-linked proteins: D1/D2, D1/cyt b559, D1/CP43.
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
Kanazawa et al. (2017). Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection. Front Plant Sci. 2017 May 3;8:719. doi: 10.3389/fpls.2017.00719.
Li et al. (2016). A Hard Day's Night: Diatoms Continue Recycling Photosystem II in the Dark. Front. Mar. Sci., 08 November 2016
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