PsbA | D1 protein of PSII, C-terminal (affinity purified)

Product no: AS05 084A

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

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  • Product Info
  • 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
    Purity: Immunogen affinity purified serum in PBS pH 7.4.
    Format: Lyophilized
    Quantity: 50 µg
    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 the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
    Tested applications: Western blot (WB)
    Recommended dilution: 1 : 15 000 (WB)
    Expected | apparent MW:

    38 | 28-30 kDa

  • Reactivity
  • Confirmed reactivity: Anabaena 7120, Arabidopsis thaliana, Artemisia annua, Arundo sp., Chlamydomonas reinhardtii, Colobanthus quitensis Kunt Bartl, Craterostigma sp., Coscinodiscus wailesii, Cynara cardunculus var altilis, Ditylum brightwellii, Glycine max, Hordeum vulgare, Lindernia sp., Miscanthus x giganteus, Marchantia polymorpha (liverwort), Nicotiana benthamiana,Panicum miliaceum, Panax ginseng, Panicum maximum, Paulinella chromatophora (amoeba), Pinus strobus,  Physcomitrium patens, Prochlorococcus sp. (surface and deep water ecotype),  Synechococcus sp. PCC 7942, Spirodela polyrhiza, Symbiodinium sp., Zea mays
    Predicted reactivity:

    Algae (brown and red), Brassica napus, Conifers, Cyanobacteria, Dictos, Cannabis sativa, Galdieria sulphuraria, Lactuca sativa, Lycopersicum esculentum, Medicago sativa, Nannochloropsis sp., Oryza sativa, Ostreococcus sp. Pisum sativum, Porphyridium purpureum, Sesamum indicum, Thalassiosira pseudonana, Zosteria marina, Vitis vinifera cellular [compartment marker] of thylakoid membrane

    Species of your interest not listed? Contact us
    Not reactive in: No confirmed exceptions from predicted reactivity are currently known
  • Application Examples
  • Application example

    western blot using affinity purified anti-PsbA antibodies
    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.
  • Additional Information
  • 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.

    Additional information (application):
    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.
  • Background
  • 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

  • Product Citations
  • Selected references: Kafri et al. (2023). Systematic identification and characterization of genes in the regulation and biogenesis of photosynthetic machinery. Cell. 2023 Dec 7;186(25):5638-5655.e25.doi: 10.1016/j.cell.2023.11.007.  
    Wada et al. (2021) Identification of a Novel Mutation Exacerbated the PSI Photoinhibition in pgr5/pgrl1 Mutants; Caution for Overestimation of the Phenotypes in Arabidopsis pgr5-1 Mutant. Cells. 2021 Oct 26;10(11):2884. doi: 10.3390/cells10112884. PMID: 34831107; PMCID: PMC8616342.
    Sorrentino et al. (2018). Performance of three cardoon cultivars in an industrial heavy metal-contaminated soil: Effects on morphology, cytology and photosynthesis. J Hazard Mater. 2018 Jun 5;351:131-137. doi: 10.1016/j.jhazmat.2018.02.044.
    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
  • Protocols
  • Agrisera Western Blot protocol and video tutorials

    Protocols to work with plant and algal protein extracts


    PsbA quantitation in plant and algal samples using Agrisera anti-PsbA antibody and PsbA protein standard

    Methodology: Plant samples are generally ground with liquid nitrogen in a mortar and pestle. The resulting powder is transferred to a plastic tube. Algal samples can be either concentrated by centrifugation or, preferably, by filtration onto glass fiber filters. Solubilization is performed in Agrisera protein extraction buffer (PEB, AS08 300) containing 0.1mg/mL PefaBloc SC (AEBSF) protease inhibitor (Roche). Disruption is most optimally obtained through flash freezing of the sample in liquid nitrogen alternated with thawing by sonication with a microtip. This process can be repeated depending on the toughness of the sample. The sample is adjusted to 50 mM dithiothreitol and heated to 70°C for 5 minutes. Samples are cooled and centrifuged briefly prior to electrophoresis.

    Optimal quantitation is achieved using moderate sample loads per gel lane, generally 0.5 to 2.5 ug total protein, depending on the abundance of the target protein.

    Electrophoresis and Immunoblotting: Once solubilized, the proteins can be separated electrophoretically in a number of systems. We obtain optimal results with the Invitrogen NuPAGE gel system using Bis-Tris 4-12% gradient gels. Proteins are separated in MES SDS running buffer according to the manufacturer’s recommendations at 200 V for 35 minutes. The gels are transferred to PVDF in the same apparatus, the SureLock XCell blot module, for 60 minutes at 30 V for a single gel or 80 minutes for a pair. Following transfer the blots are blocked with non-fat dry milk up to 10 % in TBS-T, for 1 h/RT with gentle agitation. The blot is incubated with primary antibody, usually at 1:25 000 to 1:50 000, for 1 h/RT (if extreme femtogram detection reagents are used) or in lower primary antibody dilution for less sensitivie reagents (mid picogram and lower).

    For quantitation a relatively high primary antibody: target protein ratio gives more reliable results than immunoblots at low ratios of primary antibody:target protein.

    The blot is washed extensively in TBS-T (twice briefly, once for 15 minutes and three times for five minutes). The blot is incubated with secondary antibody, for example goat anti-rabbit IgG horse radish peroxidase conjugated, AS09 602 (Agrisera), for 1h/RT. The blot is washed as above and developed with ECL detection reagents.

    Quantitation: When quantitated standards are included on the blot, the samples can be quantitated using the available software. Excellent quantitation can be obtained with images captured on the Bio-Rad Fluor-S-Max or equivalent instrument using Bio-Rad QuantityOne software. The contour tool is used to select the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample. Using above protocol linear standard curves are generated over 1-1.5 orders of magnitude range in target load. It is important to note that immunodetections usually show a strongly sigmoidal signal to load response curve, with a region of trace detection of low loads, a pseudolinear range and a region of saturated response with high loads. For immunoquantitation it is critical that the target proteins in the samples and the standard curve fall within the pseudolinear range. Our total detection range using this protocol spans over 2 orders of magnitude, but the quantifiable range is narrower.

    Recommended secondary antibodies: goat anti-rabbit HRP conjugated, goat anti-rabbit ALP conjugated

    Recommended chemiluminescent detection reagent: AgriseraECLBright

    References:

    MacKenzie et al (2005). Large reallocations of carbon, nitrogen and photosynthetic reductant among phycobilisomes, photosystems and Rubisco during light acclimation in Synechococcus elongatus are constrained in cells under low environmental inorganic carbon. Arch of Microbiol. 183: 190 - 202.
    Bouchard et al. (2006) UVB effects on the photosystem II-D1 protein of phytoplankton and natural phytoplankton communities. Photochem and Photobiol 82: 936-951.

  • Reviews:
  • Reviews
    Below, you can grade the product on a scale from 0 to 5.
    Please also provide information about species, application, dilution and obtained result for the reviewed antibody.
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    Number of reviews: (2)
    Amanda Smythers | 2020-12-03
    0 | 0
    I have used this for WBs successfully in Chlorella vulgaris, PSII enriched membranes from spinach, and Chlamydomonas reinhardtii. All were loaded with 5 ug chl a and blotted using a 1:1000 dilution before ECL detection. We've never had a problem with the antibody and it has been easy to implement in our studies.
    Peter B. Kós | 2018-08-27
    0 | 0
    Very good, reliable, high quality product. We have successfully used it in various projects, for plant (Krieger-Liszkay et al, Physiologia Plantarum 142, 2011) and cyanobacterial (Photochemistry and Photobiology, 89,2013) samples over a decade already. Also performs excellently in the project that we are currently preparing for publication. I can higly recommend using it.

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Buy 2 items of this product for 239.00 €/items
Buy 3 items of this product for 217.00 €/items
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