GLDP | Glycine decarboxylase P protein
AS20 4370 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Arabidopsis thaliana, Flaveria spp., Neurachne spp.,Oryza sativa, Zea mays
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6 µg of total protein extracted freshly from Z. mays = Zea mays, A. tha= Arabidopsis thaliana, O. sat = Oryza sativa, was freshly extracted from leaf tissue in extraction buffer (50 mM HEPES-KOH pH 7.1, 1 mM EDTA pH 8.0, 10 mM DTT, 5 mM MgCl2, 1 mM PMSF, 20 µg/ml chymostatin, 25 µg/ml aprotinin, 1X Protease Inhibitor Cocktail VI, Plant Cell (AG Scientific)). Proteins were denatured with sample buffer (1X = 62.5 mM Tris-HCl pH 6.8, 2% [v/v] SDS, 10% [v/v] glycerol, 0.01% [w/v] bromophenol blue (Sigma), 100 mM DTT), heated to 95°C in a water bath for 3 minutes and collected at 13523 x g in a bench centrifuge. 6µg of protein was loaded per well and separated on a 12% resolving gel (0.375 M Tris-HCl pH 8.8, 12% [v/v] acrylamide, 0.1% [v/v] ammonium persulfate and 0.05% [v/v] TEMED) and 5% stacking gel (0.125 M Tris-HCl pH 6.8, 5% [v/v] acrylamide, 0.1% [v/v] SDS, 0.1% [v/v] ammonium persulfate and 0.025% [v/v] TEMED). Proteins were blotted onto nitrocellulose membrane (pore size 0.45 µm) using Trans-Blot Semi-Dry Transfer Cell (Bio-Rad), and transfer was run at 25 mA per gel stack for 1½ hours. Blots were blocked in blocking solution (5% [w/v] skim milk powder in TBST (12.5 mM Tris, 137 mM NaCl, 2.7 mM KCl, 0.1% [v/v] Tween-20 detergent)) overnight at room temperature with gentle agitation. The blot was rinsed three times quickly in TBST, then incubated in the primary antibody solution at a dilution of 1:5000 in blocking solution for 1 hour at room temperature with gentle agitation. Blots were rinsed three times quickly in TBST, then washed three times for 5 minutes in TBST at room temperature with gentle agitation. Blots were then incubated in secondary antibody solution (goat anti-rabbit IgG horse radish peroxidase (Sigma) diluted 1:3000 in blocking solution) for 1 hour at room temperature with gentle agitation. The blot was washed three times for 5 minutes in TBST at room temperature with gentle agitation, then rinsed twice in TBS (12.5 mM Tris, 137 mM NaCl, 2.7 mM KCl). Blots were developed with chemiluminescent detection reagent, following manufacture's recommendations, incubating at room temperature for 1 minute and imaged with Bio-Rad ChemiDoc system. Exposure time was 40 seconds.
Courtesy of Prof. Martha Ludwig, School of Molecular Sciences, Australia
Alternative names: AtGLDP1, AtGLDP2, Glycine dehydrogenase (aminomethyl-transferring) 1, Glycine dehydrogenase (aminomethyl-transferring) 2, Glycine cleavage system P protein 1, Glycine cleavage system P protein 2, Glycine decarboxylase 1, Glycine decarboxylase 2, Glycine decarboxylase P-protein 1, Glycine decarboxylase P-protein 2.