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PsbA | D1 protein of PSII, C-terminal (rabbit antibody) (thylakoid membrane marker)

AS05 084 | Clonality: Polyclonal | Host: Rabbit | Reactivity: [global antibody] for higher plants, algae, liverwort, cyanobacteria, diatoms | cellular [compartment marker] of thylakoid membrane

PsbA | D1 protein of PSII, C-terminal (rabbit antibody) (thylakoid membrane marker) in the group Plant/Algal Antibodies / Triticum sp.  at Agrisera AB (Antibodies for research) (AS05 084)

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product information
Background

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

Immunogen

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

Host Rabbit
Clonality Polyclonal
Clone
Purity Serum
Format Lyophilized
Quantity 50 µl
Reconstitution For reconstitution add 50 µl of sterile water.
Storage Store lyophilized/reconstituted at -20°C; once reconstituted make aliquots to avoid repeated freeze-thaw cycles. Please, remember to spin tubes briefly prior to opening them to avoid any losses that might occur from lyophilized material adhering to the cap or sides of the tubes.
Tested applications ImmunoGold (IG), Western blot (WB)
Related products

AS01 016S | PsbA protein standard for a quantitative western blot
AS05 084PRE | PsbA | D1 protein of PSII, C-terminal , pre-immune serum
AS11 1786  | anti-PsbA N-terminal, rabbit antibody
AS10 704 | PsbA | D1 protein of PSII, DE-loop, rabbit antibody
AS13 2669 | PsbA | D1 protein of PSII, phosphorylated, rabbit antibody

recommended secondary antibody

Plant and algal protein extraction buffer

Secondary antibodies

Additional information

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.

application information
Recommended dilution 1: 200 (IG), 1 : 10 000 (WB)
Expected | apparent MW

38 | 28-30 kDa

Confirmed reactivity Anabaena 7120, Arabidopsis thaliana, Artemisia annua, Arundo sp., Begonia sp. , Chlamydomonas reinhardtii, Chlorella ohadii, Chromera velia, Chlorella vulgaris, Colobanthus quitensis Kunt Bartl, Coscinodiscus wailesii, Craterostigma sp., Cyanidioschyzon merolae, Cytisus cantabricus (Wilk.) Rchb. F, Dianthus caryophyllus, Ditylum brightwellii, Eucalyptus globulus, Glycine max, Halomicronema hongdechloris, Hieracium pilosella L., Hordeum vulgare, Lasallia hispanica, Lindernia sp., Marchantia polymorpha (liverwort), Medicago truncatula, Miscanthus x giganteus, Microcystis aeruginosa, Nicotiana benthamiana, Panicum miliaceum, Panax ginseng, Panicum maximum, Paulinella chromatophora (amoeba), Pheodactylum tricornutum CCAP 1055/1, Physcomitrella patens, Picea glauca, Pinus strobus, Prochlorococcus sp. (surface and deep water ecotype), Spartina alterniflora, Spirodela polyrhiza, Symbiodinium sp, Synechococcus sp. PCC 7942, Syntrichia muralis, Thalassiosira weissflogii, Triticum aestivum, Zea may, Quercus ilex
Predicted reactivity

Algae (brown and red), Conifers, Cyanobacteria, Dictos, Cannabis sativa, Lycopersicum esculentum, Medicago sativa, Pisum sativum, Sesamum indicum, cellular [compartment marker] of thylakoid membrane

Not reactive in No confirmed exceptions from predicted reactivity are currently known.
Additional information

The antibody is appropriate for detecting both, 24 kDa or the 10 kDa C-terminal fragments, whichever is generated under given treatment conditions. In our analysis we have seen both, ca. 24 kDa and ca. 10 kDa fragments from different samples, depending on treatments and isolation procedures.

Rabbit anti-PsbA antibody can detect more than one band of PsbA protein, e.g. precursor and mature protein as compare to the hen anti-PsbA antibodies AS01 016.

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.


Selected references Pao et al. (2018). Lamelloplasts and minichloroplasts in Begoniaceae: iridescence and photosynthetic functioning. J Plant Res. 2018 Mar 2. doi: 10.1007/s10265-018-1020-2. (ImmunoGold)
Muneer et al. (2018). Proteomic Analysis Reveals the Dynamic Role of Silicon in Alleviation of Hyperhydricity in Carnation Grown In Vitro. Int. J. Mol. Sci. 2018, 19(1), 50; doi:10.3390/ijms19010050.
Gao et al. (2018). Global warming interacts with ocean acidification to alter PSII function and protection in the diatom Thalassiosira weissflogii. Environmanetal and Exp. Bot. Volume 147, March 2018, Pages 95–103. Ananyev et al. (2017). Photosystem II-Cyclic Electron Flow Powers Exceptional Photoprotection and Record Growth in the Microalga Chlorella ohadii.Biochim Biophys Acta. 2017 Jul 19. pii: S0005-2728(17)30105-6. doi: 10.1016/j.bbabio.2017.07.001.
Sharwood et al. (2017). Linking photosynthesis and leaf N allocation under future elevated CO2 and climate warming in Eucalyptus globulus. J Exp Bot. 2017 Feb 1;68(5):1157-1167. doi: 10.1093/jxb/erw484.
Míguez et al. (2017). Diversity of winter photoinhibitory responses: A case study in co-occurring lichens, mosses, herbs and woody plants from subalpine environments. Physiol Plant. 2017 Feb 14. doi: 10.1111/ppl.12551.
Romanowska et al. (2017). Differences in photosynthetic responses of NADP-ME type C4 species to high light. Planta. 2017 Mar;245(3):641-657. doi: 10.1007/s00425-016-2632-1. Epub 2016 Dec 18.
Yang-Er Chen et al. (2017). Responses of photosystem II and antioxidative systems to high light and high temperature co-stress in wheat. J. of Exp. Botany, Volume 135, March 2017, Pages 45–55.

application example 1

2 µg of total protein from (1) Arabidopsis thaliana leaf extracted with Protein Extration Buffer, PEB (AS08 300), (2) Hordeum vulgare leaf extracted with PEB, (3) Chlamydomonas reinhardtii total cell extracted with PEB, (4) Synechococcus sp. 7942 total cell extracted with PEB, (5) Anabaena sp. total cell extracted with PEB were 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: 50 000 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 1:50 000 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).

 

Western blot anti-PsbA rabbit antibody

application example 2

Varying amounts of PsbA protein standard (AS01 016S) 250 fmol (1), 125 fmol (2), 62.5 fmol (3), 31.25 fmol (4), 15.625 fmol (5) and 2 µg of total protein from Med4 (6,7) extracted with Protein Extration 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 (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 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 (goat 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 TMA-6 (Lumigen) detection reagent according the manufacturers 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.

 

western blot using anti-PsbA antibodies on Med4

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