RbcL | Rubisco large subunit, form I and form II
AS03 037 | clonality: polyclonal | host: rabbit | reactivity: [global antibody] for higher plants, lichens, algae, cyanobacteria, dinoflagellates, diatoms | cellular [compartment marker] of plastid stroma in higher plants and cytoplasm in cyanobacteria
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Immunofluorescence/confocal microscopy (IF), 1: 1000 (IG), 1: 250 for images see Prins et al. (2008), detailed protocol available on request, 1: 800 (TP), 1: 5000 - 10 000 (WB)
|Expected | apparent MW||
52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 (Chlamydomonas reinhardtii)
|Confirmed reactivity||Agostis stolonifera cv. ‘Penncross’, Arabidopsis thaliana, Apium graveolens, Artemisia annua, Baculogypsina sphaerulata (benthic foraminifer), Beta vulgaris, Bienertia sinuspersici, Kandelia candel, Cicer arietinum, Chlamydomonas raudensis, Chlamydomonas reinhardtii, Colobanthus quitensis Kunt Bartl, Cyanophora paradoxa, Cylindrospermopsis raciborskii CS-505, Cynara cardunculus, Emiliana huxleyi, Euglena gracilis, Fraxinus mandshurica, Fucus vesiculosus, Glycine max, Gonyaulax polyedra, Guzmania hybrid, Heterosigma akashiwo, Hordeum vulgare, Jatropha curcas, Karenia brevis (C.C.Davis) s) G.Hansen & Ø.Moestrup (Wilson isolate), Liquidambar formosana, Malus domestica, Medicago truncatula, Micromonas pusila, Nicotiana benthamiana, Petunia hybrida cv. Mitchell, Phaeodactylum tricornutum, Physcomitrella patens, Porphyra sp., Ricinus communis, Robinia pseudoacacia, Saccharum spp., Schima superba, Stanleya pinnata, Spinacia oleracea, lichens, Symbiodinium sp., Synechococcus PCC 7942, Rhoeo discolor, Thalassiosira pseudonana, Thermosynechococcus elongatus, Prochlorococcus sp. (surface and deep water ecotype), Triticum aestivum, dinoflagellate endosymbionts (genus Symbiodinium), extreme acidophilic verrucomicrobial methanotroph Methylacidiphilum fumariolicum strain SolV, Thalassiosira punctigera, Tisochrysis lutea, Verbascum lychnitis, Vitis vinifera, Quercus ilex
|Predicted reactivity||Aalpha proteobacteria, Algae (brown and red), Dicots, Beta-proteobacteria, Conifers, Cryptomonads, Cyanobacteria (prochlorophytes), Gamma-proeobacteria, Liverworts, Monocots, Mosses, Suaeda glauca, Welwitschia|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
This antibody was used in:
Immunocytochemical staining of diatoms according to Schmid (2003) J Phycol 39: 139-153 and Wordemann et al. (1986) J Cell Biol 102: 1688-1698.
Immunofluorescence Dreier et al. (2012). FEMS Microbial Ecol., March 2012.
Western blot and tissue printing during a student course Ma et al. (2009).
|Selected references||Ḱim et al. (2017). Effect of cell cycle arrest on intermediate metabolism in the marine diatom Phaeodactylum tricornutum. Proc Natl Acad Sci U S A. 2017 Sep 19;114(38):E8007-E8016. doi: 10.1073/pnas.1711642114.
Arena et al. (2017). Eco-physiological and Antioxidant Responses of Holm Oak (Quercus ilex L.) Leaves to Cd and Pb. Water, Air, & Soil Pollution December 2017, 228:459.
Jespersen et al. (2017). Metabolic Effects of Acibenzolar-S-Methyl for Improving Heat or Drought Stress in Creeping Bentgrass. Front Plant Sci. 2017 Jul 11;8:1224. doi: 10.3389/fpls.2017.01224. eCollection 2017. (western blot, Agostis stolonifera cv. ‘Penncross’)
Neto et al. (2017). Cyclic electron flow, NPQ and photorespiration are crucial for the establishment of young plants of Ricinus communis and Jatropha curcas exposed to drought. Plant Biol (Stuttg). 2017 Apr 12. doi: 10.1111/plb.12573. (Jatropha curcas and Ricinus communis, western blot)
Ribeiro et al. (2017). Increased sink strength offsets the inhibitory effect of sucrose on sugarcane photosynthesis. J Plant Physiol. 2017 Jan;208:61-69. doi: 10.1016/j.jplph.2016.11.005.
Baumgart et al. (2017). Heterologous expression of the Halothiobacillus neapolitanus carboxysomal gene cluster in Corynebacterium glutamicum. J Biotechnol. 2017 Mar 27. pii: S0168-1656(17)30124-4. doi: 10.1016/j.jbiotec.2017.03.019.
Kolesinski et al. (2017). Is RAF1 protein from Synechocystis sp. PCC 6803 really needed in the cyanobacterial Rubisco assembly process? Photosynth Res. 2017 Jan 20. doi: 10.1007/s11120-017-0336-4.
Castiglia et al. (2016). High-level expression of thermostable cellulolytic enzymes in tobacco transplastomic plants and their use in hydrolysis of an industrially pretreated Arundo donax L. biomass.Biotechnol Biofuels. 2016 Jul 22;9:154. doi: 10.1186/s13068-016-0569-z. eCollection 2016.
Meng et al. (2016). Physiological and proteomic responses to salt stress in chloroplasts of diploid and tetraploid black locust (Robinia pseudoacacia L.). Sci Rep. 2016 Mar 15;6:23098. doi: 10.1038/srep23098
Heinnickel et al. (2016). Tetratricopeptide repeat protein protects photosystem I from oxidative disruption during assembly. Proc Natl Acad Sci U S A. 2016 Mar 8;113(10):2774-9. doi: 10.1073/pnas.1524040113
Young et al. (2015). Antarctic phytoplankton down-regulate their carbon-concentrating mechanisms under high CO2 with no change in growth rates. Marine Ecology Progress Series 532:13-28.
Li at al. (2015). Salt stress response of membrane proteome of sugar beet monosomic addition line M14. J Proteomics. 2015 Apr 3. pii: S1874-3919(15)00109-8. doi: 10.1016/j.jprot.2015.03.025.
Krasuska et al. (2015). Switch from heterotrophy to autotrophy of apple cotyledons depends on NO signal. Planta. 2015 Jul 18.
Janeczko et al. (2015). Disturbances in production of progesterone and their implications in plant studies. Steroids. 2015 Feb 9. pii: S0039-128X(15)00054-9. doi: 10.1016/j.steroids.2015.01.025.
Kolesinski et al. (2014). Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro. FEBS J. 2014 Jul 12. doi: 10.1111/febs.12928
Pandey and Pandey-Rai (2014). Modulations of physiological responses and possible involvement of defense-related secondary metabolites in acclimation of Artemisia annua L. against short-term UV-B radiation. Planta. 2014 Jul 15.
Liang et al. (2014). Cyanophycin mediates the accumulation and storage of fixed carbon in non-heterocystous filamentous cyanobacteria from coniform mats. PLoS One. 2014 Feb 7;9(2):e88142. doi: 10.1371/journal.pone.0088142. eCollection 2014. (immunogold)
Mayfield et al. (2014). Rubisco Expression in the Dinoflagellate Symbiodinium sp. Is Influenced by Both Photoperiod and Endosymbiotic Lifestyle. Mar Biotechnol, Jan 22.
0.25 µg of chlorophyl a/lane from Spinacia oleracea (1), Synechococcus PCC 7942 (2), Cyanophora paradoxa (3), Heterosigma akashiwo (4), Thalassiosira pseudonana (5), Euglena gracilis (6), Micromonas pusilla (7), Chlamydomonas reinhardtii (8), Porphyra sp (9), Gonyaulax polyedra (10), Emiliania huxleyi (11) extracted with PEB (AS08 300), were separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to nitrocellulose. Filters were blocked 1h with 2% low-fat milk powder in TBS-T (0.1% TWEEN 20) and probed with anti-RbcL antibody (AS03 037, 1:50 000, 1h) and secondary anti-rabbit (1:20000, 1 h) antibody (HRP conjugated, recommended secondary antibody AS09 602) in TBS-T containing 2% low fat milk powder. Antibody incubations were followed by washings in TBS-T. All steps were performed at RT with agitation. Blots were developed for 5 min with ECL Advance detection reagent according the manufacturers instructions (GE Healthcare). Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).
1 µg of chlorophyll from Cryptophyte samples (1,2) and 1 µg of chlorophyll (3) or 10 µg of total protein (4) from Arabidopsis thaliana leaves extracted either with 2ml of 100 mM TrisHCl, 50 mM EDTA, 250 mM NaCl, 0.05% SDS (Sample 1) or 10 mL of 50 mM Hepes-KOH (pH 7.8), 330 mM sorbitol, 10 m EDTA, 5 mM NaCl, 5 mM MgCl2, 5 mM sodium ascorbate and 0.2% BSA (Sample 2). Samples were denatured with 1:1 Amersham WB Loading Bufferv at 70C for 10 min and were separated on pre-casted 13.5% Amersham WB gel and blotted for 30 min to Amersham WB PVDF using wet transfer. Blots were blocked with 2% Amersham ECL Blocking Agent for 1h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 (rabbit anti-Rubisco AS03 037) for 1.5 h at RT 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 RT with agitation. Membrane was cut in half and left part was incubated in anti-rabbit DyLight® 550 secondary antibody from Agrisera (AS11 1782) diluted to 1:2 000 in TBST for 1h at RT with agitation. The blot was scanned using Cy3 channel of Amersham WB System.
Courtesy Dr. Małgorzata Wessels, Agrisera
2 µg of total protein from various plant extracts (1-5) extracted with PEB (AS08 300) separated on 4-12% NuPage (Invitrogen) LDS-PAGE and blotted 1h to PVDF. Markers MagicMarks (Invitrogen) (M) and Rubisco protein standard (AS01 017S) at 0.0625 pmol, 0.125 pmol, 0.25 pmol.
Following standard western blot procedure this image has been obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad). The contour tool of the software is used to the area for quantitation and the values are background subtracted to give an adjusted volume in counts for each standard and sample.
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