Protein samples were denatured by heating in the presence of loading buffer (100 mm Tris–HCl, pH 8, 25% [v/v] glycerol, 1% [w/v] SDS, 10% [v/v] β‐mercaptoethanol, and 0.05% [w/v] bromophenol blue). Denatured proteins (10 μg for leaf extracts and 50 μg for root extracts) were separated by SDS‐PAGE in a Mini‐Protean® III‐2D Cell (Bio‐Rad, Hercules, CA, USA) and electroblotted onto a PVDF or nitrocellulose membrane in a semidry transfer blot system (Bio‐Rad). Polyclonal antibodies against native C4‐photosynthetic PEPC from sorghum leaves (anti‐C4 PTPC) were prepared as described in Pacquit et al. (1995 (link)). These antibodies recognize both photosynthetic and non‐photosynthetic PEPCs (Ruiz‐Ballesta et al., 2016 (link)). Immunolabeled proteins were detected by a chemiluminescence detection system (SuperSignal West Pico Rabbit IgGs; Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions in an Amersham Imager 600 (GE‐Healthcare, Chicago, IL, USA). The signal intensities were quantified using Image Studio™ Lite software (LI‐COR, Lincoln, NE, USA).
Mini protean 3 2d cell
Manufactured by Bio-Rad
Sourced in United States
The Mini-Protean® III-2D Cell is a laboratory equipment used for performing two-dimensional (2D) gel electrophoresis. It is designed for the separation and analysis of complex protein mixtures. The device allows for the separation of proteins based on their isoelectric point and molecular weight.
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Lab products found in correlation
Semidry transfer blot system, by Bio-Rad (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Supersignal west pico rabbit iggs, by Thermo Fisher Scientific (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Coomassie blue r 250, by Merck Group (1 mentions)
Semi dry transfer blot apparatus, by Bio-Rad (1 mentions)
2 protocols using mini protean 3 2d cell
1
SDS-PAGE and Immunoblotting of Plant PEPCs
2
SDS-PAGE and Immunoblotting Protein Analysis
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Protein samples were subjected to SDS–PAGE (10% [w/v] acrylamide) according to the method of Laemmli (Laemmli, 1970 (link)) at room temperature for 2 h at 100 V in a Mini-Protean ®III-2D cell (Bio-Rad). After electrophoresis, proteins on the gels were stained with Coomassie Blue R-250 or electroblotted onto a nitrocellulose membrane (N-8017, Sigma) at 10 V (5.5 mA cm-2) for 30 min in a semidry transfer blot apparatus (Bio-Rad Laboratories). Membranes were blocked in Tris-buffered saline (20 mM Tris–HCl and 0.15 mM NaCl [pH 7.5]) containing 5% (w/v) powdered milk, and bands were immunochemically labeled by overnight incubation of the membrane at 4°C in 20 ml of Tris-buffered saline containing specific antibodies. Subsequent detection was performed using a horseradish peroxidase conjugated antibody (Bio-Rad) by a peroxidase assay (Figure 1 , 2 , 3 , 5 ) or by a chemiluminescence detection system (Super Signal West Dura Signal; ThermoFisher, Waltham, MA, United States) according to the manufacturer’s instructions (Figure 4 ).
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