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14 protocols using anti rbcl

1

Cyanobacterial Protein Extraction and Western Blotting

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The protein extracts from cyanobacteria cells were prepared according to the method described by Nimura et al. (2001) (link) and were separated by SDS-PAGE on 12% polyacrylamide gels at 150 V for 90–100 min in electrophoresis buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS). The gels were blotted onto 0.2 μm PVDF membranes (Bio-Rad, Hercules, CA, United States) at 2.5 A for 10 min using a Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were blocked for 30 min with 5% skimmed milk in TBS-T [50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20], washed, and soaked in TBS-t for 2 h at room temperature with anti-GFP (MBL, Tokyo, Japan), anti-RpoD1 rabbit antiserum (Seki et al., 2007 (link)), or anti-RbcL (Agrisera, Vännäs, Sweden) as primary antibodies. After washing, the membranes were soaked in TBS-t for 30 min at room temperature with HRP-conjugated anti-mouse (GE Healthcare, Chicago, IL, United States) or anti-rabbit antibody (GE Healthcare) as secondary antibodies. Chemiluminescence was detected with the LumiGLO Chemiluminescent Substrate KPL using ChemiDoc XRS Plus (Bio-Rad).
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2

Protein Analysis via SDS-PAGE and Immunoblot

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SDS-PAGE and immunoblot
analysis were performed as described previously.23 (link),31 (link),52 (link) Protein concentrations were quantified by
the Bradford method.53 (link) Anti-RbcL (1:10,000
dilution, Agrisera, Sweden), anti-CsoS1 from H. neapolitanus (1:5000 dilution, Agrisera, Sweden), and anti-HisTag (Invitrogen,
USA) antibodies and horseradish peroxidase-conjugated goat antirabbit
immunoglobulin G secondary antibody were used for immunoblot analysis
and imaged on an Image Quant LAS 4000 platform (GE Healthcare Life
Sciences, USA).
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3

Immunogold Labeling of Cryo-Fixed Samples

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For immunogold labeling, cryo-fixed and freeze-substituted samples embedded into LR White were cut into 100-nm-thin sections. Nonspecific antibody binding was reduced by incubation of samples in blocking buffer [phosphate-buffered saline–Tween 20 (PBST) containing 2% bovine serum albumin and 0.1% fish gelatin; Sigma-Aldrich] for 1 hour at room temperature. Antigens were detected by incubation in blocking buffer containing anti-dsRed (1:100 dilution, mouse; ChromoTek GmbH, Planegg-Martinsried, Germany), anti-ClpP (1:200, rabbit; provided by A. Clarke, University of Gothenburg, Sweden), anti-RbcL (1:200, rabbit; Agrisera, Vännäs, Sweden), or anti-GLN2 (1:200, rabbit; Agrisera) antibodies for 1 hour at room temperature. Excess antibodies were removed by six rinses with PBST buffer for 3 min each. Following hybridization to sections, bound primary antibodies were detected by incubation for 1 hour in blocking buffer containing 10 nm (for mouse; Cell Signaling Technology, Danvers, MA) or 25 nm colloidal gold-labeled secondary antibodies (goat anti-rabbit antibody, 1:20; AURION, Wageningen, The Netherlands). After six rinses with PBST and three rinses with double-distilled water for 3 min each, samples were contrasted with aqueous 2% uranyl acetate for 12 min and Reynolds’ lead citrate for 7 min.
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4

Nuclear Protein Detection and Analysis

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The nuclear proteins were extracted as previously described (Choudhary et al., 2009 ). The protein samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and analysed by protein blotting with anti-OsNMD3, anti-GFP, anti-Histone3, anti-RbcL, anti-hsRPL4, and anti-hsRPS14 antibodies, respectively. The anti-Histone3 and anti-RbcL were purchased from Agrisera. The secondary antibody, horseradish peroxidase-conjugated anti-rabbit IgG, was used for staining.
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5

Chloroplast Protein Immunoblotting Analysis

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Crude chloroplast proteins were loaded onto a sodium dodecyl sulfate–polyacrylamide gel on an equal Chl basis. Samples of 0.5 µg Chl proteins from the crude chloroplast fraction were used for immunoblotting. Immunoblotting was performed with anti-ChlB (Fujita et al. 1996 (link)), anti-RbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) (Agrisera, Vännäs, Sweden), and anti-L-POR (Agrisera) antibodies. Signals were detected with an ECL Plus Western Blotting Detection Kit (GE Healthcare UK Ltd, Buckinghamshire, UK) and visualized with an LAS3000 chemiluminescence analyzer (Fuji Film, Tokyo, Japan). The results presented represent two independent experiments.
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6

Protein Expression and Phosphorylation Analysis

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Protein expression and phosphorylation were assessed using standard Western blotting protocols described by Chen and colleagues [63]. Blots were probed with rabbit polyclonal anti-AtpB, anti-PsaD, anti-PsbC, anti-RA, anti-RbcL, anti-RbcS, and anti-UGPase antibodies (Agrisera Antibodies, Vännäs, Sweden) and an anti-plant-actin rabbit polyclonal antibody (EasyBio, Beijing, China). The rabbit polyclonal anti-PPDK and anti-PEPCK antibodies were prepared by our laboratory.
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7

Isolation and Analysis of Chloroplast Proteins

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The protocol for isolating intact chloroplasts from 21‐day‐old leaves was described previously (Sun, Chen, Lin, & Li, 2001 (link)). [35S]‐methionine‐labeled proteins were synthesized through in vitro transcription and in vitro translation of wheat germ extracts (TnT Quick Coupled Transcription/Translation System, Promega) according to the manufacturer's specifications. Labeled proteins were separated by 10% SDS‐PAGE and quantified with a PhosphorImager SP system (Molecular Dynamics). For immunoblot analysis, 25 μg of chloroplast proteins were fractionated by 4%–12% SDS‐PAGE, blotted onto polyvinylidene difluoride membrane, and hybridized with anti‐Toc33, anti‐RbcL, anti‐RbcS, or anti‐GS2 antiserum (Agrisera, Sweden) according to manufacturer's suggestion. Western blot signals were quantified using a LAS 4000 image analyzer (GE Healthcare) and calibrated based on the signals for Col‐0.
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8

Synechocystis Protein Extraction and SDS-PAGE Analysis

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The wild-type and mutant cultures of Synechocystis were diluted to OD750 0.6 and cultivated in an L/D cycle. Five milliliter of WT and mutant cultures of Synechocystis were harvested by centrifugation at 4,000 ×g for 2 min at 4°C. After the pellet was resuspended in PBS buffer (8.6 mM Na2PO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH 7.5), the cells were disrupted in a cell mill using glass beads. The crude extract was obtained by centrifugation at 500 ×g for 5 min at 4°C, and different protein amounts of the crude extract were subjected to SDS-PAGE. The immunodetection was performed using anti-RbcL (Agrisera, Sweden) and secondary anti-rabbit (Thermo Fisher Scientific Inc., USA) antibodies.
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9

Protein Blotting and Detection Protocol

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After SDS-PAGE separation, proteins were transferred onto a 0.2 µm polyvinylidene difluoride (PVDF) membrane using a Trans-Blot® Turbo transfer system (Bio-Rad Laboratories) for 7 min at 25 V. Membranes were blocked with 5% skimmed milk powder (w/v) in phosphate buffered saline with 0.05% Tween-20 (PBS-T) buffer for 60 min at room temperature and incubated in a primary antibody solution at 4 °C overnight. Various primary antibodies were used with the following dilutions: Anti-HA 1:1000 (Sigma-Aldrich), Anti-RbcL 1:5000 (Agrisera) and Anti-PsbA 1:8000 (Agrisera). The membrane was then washed with PBS-T and incubated for 60 min at room temperature with an Anti-Rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Dako) at a dilution of 1:5000. After more washing steps, the HRP signal was developed using SuperSignal West Dura Substrate (Thermo Scientific) and detected with a ChemiDoc MP imaging system (Bio-Rad Laboratories).
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10

Antibody Characterization for Protein Detection

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Anti-Myc (Cat# M4439 RRID: AB_439694) and anti-FLAG (Cat# F3165 RRID: AB_259529) were purchased from Sigma-Aldrich (USA). Anti-H3 (Cat# AS10 710 RRID: AB_10750790), anti-RbcL (Cat# AS03 037–200 RRID: AB_2175288) and anti-14-3-3 (Cat# AS12 2119 RRID: AB_2619715) were from Agrisera (Sweden). Anti-GFP (Cat# MMS-118P-200 RRID: AB_10063778) was from Covance (USA).
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