RbcL | Rubisco large subunit, form I
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|Recommended dilution||(IL) tested on a grass species, formaldehyde-fixed and paraffin-embedded tissue following the protocol from Gonzalez et al. (1998) Plant Physiol. V. 116. 1 : 10 000-1 : 20 000, 2 ĩg of total cellular protein, (WB)|
|Expected | apparent MW||
52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 kDa (Chlamydomonas reinhardtii)
|Confirmed reactivity||Arabidopsis thaliana, Lobaria pulmonaria, Medicago sativa, mixed phytoplankton, Pinus strobus, Pisum sativum, Solanum tuberosum, Spartina alterniflora, Spinacia oleracea, Synechococcus sp. PCC7842, Thiobacillus sp. Ulmus sp.|
|Predicted reactivity||Algae, Dicots, Conifers, Liverworts, Mosses, Prochlorophytes, Welwitschia|
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
This antibody detects RbcL protein from 102.6 fmoles and has been used as a control to ensure adequate permeabilization and fixation of toxic cyanobacterial cells in immunolabeling experiments (method based on: Orellana & Perry (1995) J Phycol 31: 785-794).
|Selected references||Robert et al. (2015). Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein. Plant Biotechnol J. 2015 Aug 19. doi: 10.1111/pbi.12452.
Morash et al. (2007). Macromolecular dynamics of the photosynthetic system over a seasonal developmental progression in Spartina alterniflora. Canadian J. of Botany, 2007, 85(5): 476-483, 10.1139/B07-043.
MacKenzie et al. (2005). Inorganic carbon acclimation in Synechococcus elongatus alters the dynamics of macromolecular pooks and photosynthetic fluxes in response to increased light. Photosynt Research 85: 341-357.
Schofield et al. (2003). Changes in macromolecular allocation in nondividins algal symbionts allow for photosynthetic acclimation in the lichen Lobaria pulmonaria. New Phytol 159: 709-718.
1 µg of total protein from samples such as Arabidopsis thaliana leaf (1) , Hordeum vulgare leaf (2), Zea mays leaf (3), Chlamydomonas reinhardtii 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 (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 (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS10 1489, 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.
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