Proteins were separated using NuPAGE 4–12% Bis-Tris Gels (Invitrogen, Carlsbad, CA) and visualized after Western blotting. Blots were blocked with 5% nonfat dried milk. Primary antibodies used to detect proteins were affinity-purified rabbit anti-LIN-41(203–420) R214 antibody (100 ng/ml) (Spike et al. 2014a (link)) and rabbit anti-GFP NB600-308 antibody (250 ng/ml) (Novus Biologicals). The secondary antibody used for Western blots was peroxidase-conjugated donkey anti-rabbit antibody (1:30,000) (Jackson ImmunoResearch). Detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
Peroxidase conjugated donkey anti rabbit antibody
Manufactured by Jackson ImmunoResearch
Peroxidase-conjugated donkey anti-rabbit antibody is a secondary antibody that binds to primary rabbit antibodies. The peroxidase enzyme is conjugated to the antibody, enabling detection and quantification of target proteins in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit anti gfp nb600 308 antibody, by Novus Biologicals (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Pageblue protein staining solution, by Thermo Fisher Scientific (1 mentions)
Con a biotin, by Vector Laboratories (1 mentions)
Vectastain abc hrp kit, by Vector Laboratories (1 mentions)
4 protocols using peroxidase conjugated donkey anti rabbit antibody
1
Western Blot Analysis of Proteins
2
Generating Antibodies against SACY-1 Protein
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sacy-1 cDNA sequences were cloned into the E. coli expression vector pMal-c2 to create an inducible fusion protein wherein maltose binding protein was fused to amino acids 411–578 of SACY-1 (MBP::SACY-1(411–578)). MBP::SACY-1(411–578) was column- and gel-purified and used to immunize rabbits. Immunizations and sera collection were performed using standard protocols (Cocalico Biologicals, Reamstown, PA). Rabbit antibody (R217) was affinity purified, and was suitable for use in western blots with partially purified SACY-1 preparations. Hybridoma cell lines producing anti-GFP monoclonal antibodies 12A6 and 4C9 (Sanchez et al. 2014 (link)) were obtained from the Developmental Studies Hybridoma Bank and prepared as described (Tsukamoto et al. 2017 (link)). Proteins were separated using NuPAGE 4–12% Bis-Tris gels (Invitrogen, Carlsbad, CA) and visualized after western blotting. Blots were blocked with 5% nonfat dried milk. Primary antibodies used to detect proteins were affinity-purified rabbit anti-SACY-1(411–578) R217 antibody (100 ng/ml) and rabbit anti-GFP NB600-308 antibody (250 ng/ml; Novus Biologicals). The secondary antibody used for western blots was peroxidase-conjugated donkey anti-rabbit antibody (1:30,000; Jackson ImmunoResearch). Detection was performed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
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3
Quantifying Protein Carbonyl Modifications
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Protein carbonyls were quantified by derivatization with 2,4-dinitrophenyl hydrazine (DNPH). Subcellular fractions containing 8 µg protein were adjusted to identical volume with buffer A, supplemented with 0.5 volumes of buffer B (180 mM TRIS pH 7.4, 30% sucrose, 6% SDS), mixed, and incubated with 0.5 volumes of freshly prepared DNPH derivatization solution (10 mM DNPH in 2 M HCl) for 20 min at room temperature. After neutralization with 0.33 volumes of 3 M TRIS base, the samples were supplemented with 0.33 volumes of loading buffer (200 mM TRIS pH 6.8, 40% glycerol, 20% β-mercaptoethanol, 8% SDS, 0.04% bromophenol blue), separated by SDS-PAGE in a 10% gel (acrylamide:bis-acrylamide 29:1) and transferred onto nitrocellulose membranes following standard protocols. After 1 h incubation with 5% fat-free dry milk in TBST, the membranes were probed with anti-2,4-dinitrophenyl (anti-DNP) antibody (1:1000; from Invitrogen) in TBST at 4 °C overnight. The membranes were washed three times with TBST, incubated for 1.5 h with peroxidase-conjugated donkey anti-rabbit antibody (1:10,000; from Jackson Immunoresearch) at room temperature, washed three times with TBST again, and visualized by enhanced chemiluminescence. Ponceau staining (0.1% Ponceau S in 5% acetic acid) of the blots was performed as loading control.
4
Protein Sample Analysis via SDS-PAGE and Blotting
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Protein samples were separated via SDS-PAGE and either stained with PageBlue Protein Staining Solution (Thermo Fisher Scientific) or used for Western blot or ConA lectin blot analysis. For Western blots, a polyclonal rabbit anti-Strep (IBA) and a polyclonal peroxidase-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch) were used as a primary and secondary antibody, respectively. For ConA lectin blots, high-mannose N-glycosylated proteins were detected employing ConA-biotin (Galab) and the VECTASTAIN ABC-HRP Kit (Vector Laboratories).
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