Anti-RPE | Ribulose-5-phosphate-3-epimerase, chloroplastic

Samples: 
1 - 10 µg of Spinacia oleracea total cell extract
2 - 10 µg of Solanum lycopersicum total cell extract
3 - 10 µg of Cucumis sativus total cell extract
4 - 10 µg of Solanum tuberosum total cell extract
5 - 10 µg of Rubisco protein standard (SIGMA)
6 - 10 µg of BSA (negative control)

10 µg/well of total protein extracted from frozen leaves from Spinacia oleracea, Solanum lycopersicum, Cucumis sativus, Solanum tuberosum. Leaves were pressed into a juice using an Angel Juicer (5500). Samples were normalised to 1mg/mL based on a Bradford assay, using ddH20 (pH 7.5) as dilutant. Samples were mixed in a 3:1 (v/v) ratio with Laemmli loading buffer containing 8% SDS, 1% bromophenol blue, 10 mM EDTA, 250 mM Tris-HCl (pH 6.8), 40% glycerol, and 20% β-mercaptoethanol. Mixtures were heated at 95 °C for 5 minutes, briefly centrifuged (4000xg, 5mins) and loaded onto 15-well 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Bio-Rad, 4561096) using a Bio-Rad Mini-Protein cell (Bio-Rad, Hercules, USA). Protein transfer was carried out on nitrocellulose (pore size of 0.45 µm - Nitrocellulose Membrane, Roll (Bio-Rad) (#1620115) & Cytiva Whatman™ Grade GB003 Blotting Paper (11959257)), using: semi-dry or dry transfer (TGX Turbo Setting). Blot was blocked with 5 % milk (in 1x PBS) 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 1h/RT with agitation in 5% milk (in 1x PBS). 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 PBS-T at RT with agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated) diluted to 1:25000 in 5% milk (in 1x PBS) for 1h/RT with agitation. The blot was washed as above and developed with a following chemiluminescent detection reagent: SuperSignal™ West Pico PLUS Chemiluminescent Substrate. Exposure on the Image Quant LAS 500 instrument using the ‘Auto’ chemiluminescence option.

Note: For detection of RPE in different species, protein load/well and primary antibody dilution and incubation time must be optimized.

Courtesy of Jack Pattinson, IAFRI PhD Student, Molecular Life Sciences (MLS), School of Natural and Environmental Sciences Devonshire Building, Newcastle University, United Kingdom

Alternative names: R5P3E, Pentose-5-phosphate 3-epimerase, Protein EMBRYO DEFECTIVE 2728