RbcL | Rubisco large subunit, form I (chicken)
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KLH-conjugated synthetic peptide derived from all known plant,algal and cyanobacterial RbcL (Rubisco large subunit of Rubisco Form I) sequences, including Arabidopsis thaliana UniProt: O03042, TAIR: AtCg00490, Synechococcus sp. Q3ALL1
52.7 kDa (Arabidopsis thaliana), 52.5 kDa (cyanobacteria), 52.3 kDa (Chlamydomonas reinhardtii)
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, 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 chemiluminescent 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.
Peptide target used to elicit this antibody is not conserved in type II Rubisco found in dinoflagellates and some photosynthetic bacteria.
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).
Antibody has been used in immunolabelling of intact cyanobacterial cells fixed with ethanol using a secondary anti-IgY antibody conjugated with a fluorochrome.
For Rubisco quantification using quantitative western blot technique, anti-RbcL antibody, (AS03 037) combined with Rubisco ready to use standard (AS01 017) is recommended.
Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the rate-limiting step of CO2 fixation in photosynthesis. It is one of the most abundant proteins on Earth and its homology has been demonstrated from purple bacteria to flowering plants.
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