Anti-RbcS | Rubisco small subunit (SSU)

Algae, Camellia oleifera, Erythranthe guttata, Flaveria bidentis, Flaveria sonorensis, Glycine max, L, Marchantia paleacea, Musa acuminata, Nicotiana benthamiana, Oryza sativa, Petunia hybrida, Polianthes tuberosa, Populus deltoides, Triticum aestivum, Solanum melongena, Solanum tuberosum, Zea mays

Application example



2 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), extracted with Agrisera PEB extraction buffer (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 (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 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 25 seconds.

Courtesy of Mayura Manerkar, Mount Alison University, Canada

Rubisco catalyzes the rate-limiting step of CO2 fixation in photosynthesis. This enzyme contains two subunits, each present in eight copies. In plants and green algae, 55-kD large subunit is coded by the chloroplast rbcL gene, and the 15-kD small subunit is coded by a family of nuclear RbcS genes.