Purified WT and mutant GlnRS samples (2.5 μg) were analyzed on Mini-Protean TGX Stain-Free SDS-PA gels. Specific bands were excised from the gel and further analyzed by mass spectrometry at the core facility, the Research Resource Center, University of Illinois at Chicago (Chicago, IL). The western blot analyses were performed on samples under reducing conditions. Following separation on either SDS-PA or native 4–16% Bis-Tris Native PA (Life Technologies) gels, the protein bands were transferred onto a PVDF membrane. The GlnRS bands were detected using anti-GlnRS mouse antibody (Santa Cruz Biotechnology) and anti-mouse IgG coupled with horseradish peroxidase (Qiagen), while the GroEL bands were detected using rabbit anti-GroEL antibody and anti-rabbit IgG horseradish peroxidase conjugate (Sigma Aldrich). An enhanced chemiluminescence (ECL) substrate kit (Amersham) was used for detection following standard manufacturer's protocols.
Anti rabbit igg horseradish peroxidase conjugate
Manufactured by Merck Group
Sourced in United States
Anti-rabbit IgG–horseradish peroxidase conjugate is a lab equipment product that functions as a detection reagent. It consists of horseradish peroxidase enzyme covalently attached to anti-rabbit immunoglobulin G (IgG) antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Ecl substrate kit, by Cytiva (1 mentions)
Fe sod, by Agrisera (1 mentions)
Mn sod, by Agrisera (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Hyperfilm, by GE Healthcare (1 mentions)
Mouse anti his, by Merck Group (1 mentions)
Anti human prdx1 antibody, by Abcam (1 mentions)
5 protocols using anti rabbit igg horseradish peroxidase conjugate
1
Proteomic Analysis of Glutaminyl-tRNA Synthetase
2
Antioxidant Enzyme Profiling in Plants
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The procedure of protein extraction, electrophoresis, electrotransfer, and Western blot were the same as described in Section 4.3.4 . The antibodies directed against particular enzymes were commercially available and produced by Agrisera® company (Vännäs, Sweden): APX (AS08368), CAT (AS09501), Fe-SOD (AS06125), Mn-SOD (AS09524), Cu/Zn-SOD (AS10652), GR (AS06181), and GPX (AS04055). The primary antibody was diluted 1:4000, and the antigen–antibody complexes were detected using a secondary anti-rabbit IgG–horseradish peroxidase conjugate diluted 1:20000 (Sigma, St. Louis, MO, USA, currently member of Merck Group, Dormstadt, Germany). The measurements were carried out in three replicates at each time-point.
3
Quantifying Chloroplast Protein pFBA by Western Blotting
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To estimate a pFBA protein accumulation level a Western blot analysis was performed, with the antibody directed against pFBA. The antibody was produced by Agrisera® company1 using a rabbit host immunized with a highly specific pFBA 15 amino acid peptide (TFEVAQKVWAETFYY). The peptide was selected on the basis of comparison between pFBA sequence of the two analyzed introgression forms, and available in database sequence of a cytosolic enzyme of B. distachyon (XM_003564823.1) to avoid a cross-reaction of the anti-pFBA antibody with a cytosolic FBA. The detailed protocols for a protein extraction and Western blotting were as described by Pawłowicz et al. (2012) (link). Briefly, 10 μg of chloroplast proteins from each time-point and introgression form in three biological replicates and standard samples were separated by 12% SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes (Bio-Rad). Immunodetection was performed with a rabbit polyclonal antibody (diluted 1:4000; Agrisera). The antigen–antibody complexes were detected using a chemiluminescent detection system with a secondary anti-rabbit IgG–horseradish peroxidase conjugate (diluted 1:20 000; Sigma) and a chemiluminescent substrate (Westar Supernova – Cyanogen) and the products intensities were estimated using ImageJ software.
4
Western Blot Analysis of Cor14b Protein
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The procedure of protein extraction, electrophoresis, electrotransfer, and Western blot were the same as described in Section 4.3.4 . The target protein was detected using a specific polyclonal antibody produced by Agrisera® company (Vännäs, Sweden), using a rabbit host, immunized with a highly specific amino acid peptide–GKAQEATEGAVEGAKTC. The peptide was designed using amino acid sequence of Cor14b obtained on the base of the cloned cDNA of cor14b. The primary antibody was diluted 1:5000, and the antigen–antibody complexes were detected using a secondary anti-rabbit IgG–horseradish peroxidase conjugate diluted 1:20,000 (Sigma, St. Louis, MO, USA, currently member of Merck Group, Dormstadt, Germany). To normalize the measurements of protein band intensities between the different Western blots, the measurements from a single membrane were divided by the mean of two standard samples separated on this blot and further multiplied by the mean of standards from all the blots. Normalization was performed to minimize the differences occurred between blots during the separated procedures. The measurements were carried out in three replicates at each time-point.
5
Western Blot Protein Detection Protocol
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The protein samples were applied to 10–15 % acrylamide SDS gels and transferred to nitrocellulose membranes (Merck Millipore, Darmstadt, Germany) by electroblotting. After blocking in 5 % non-fat milk in PBS containing 0.05 % Tween 20, membranes were probed with various primary antibodies: polyclonal rabbit anti-human Prdx1 antibody (Abcam), mouse anti-His (Sigma-Aldrich), followed by detection with anti-rabbit-IgG-horse-radish peroxidase conjugate (Sigma-Aldrich) or anti-mouse IgG horseradish peroxidase conjugate (Stratagene, Agilent Technologies, Santa Clara, CA. USA). Western blots were developed using advanced chemiluminescence (ECL) reagents (GE Healthcare, Amersham, UK) and exposed to autoradiography using Hyper-film (GE Healthcare).
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