A total of 41 centromere proteins were cloned into pEFs vector or pcDNA3.4 vector as a centromere protein library, combined with the streptavidin-binding peptide tag and green fluorescent protein (GFP) at the N-terminus and expressed by 293T cells. Most of them were cotransfected to construct subcomplexes according to the molecular structure of the centromere.8 9 21–23 (link) Antigens were purified by streptavidin beads and electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by silver staining (Aproscience, Tokushima, Japan) and western blotting with anti-human GFP antibody (unconjugated, 1GFP63, BioLegend, California, USA) and horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (GE Healthcare, Buckinghamshire, UK) (see online supplemental figure S1 ). Nucleic acid of CENP-E was purchased from Kazusa DNA Research Institute (Chiba, Japan). Other detailed methods were described previously.17 (link)
Horseradish peroxidase conjugated sheep anti mouse igg antibody
Manufactured by GE Healthcare
Sourced in United States
Horseradish peroxidase-conjugated sheep anti-mouse IgG antibody is a laboratory reagent used to detect the presence of mouse immunoglobulin G (IgG) in biological samples. The antibody is conjugated with the enzyme horseradish peroxidase, which enables colorimetric or chemiluminescent detection when the appropriate substrate is added.
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Lab products found in correlation
Pierce ecl western blotting substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosphatase inhibitor, by Merck Group (1 mentions)
Smad2 3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Phospho smad2 3, by Cell Signaling Technology (1 mentions)
Igf 1rβ, by Cell Signaling Technology (1 mentions)
Phospho igf1rβ, by Cell Signaling Technology (1 mentions)
2 protocols using horseradish peroxidase conjugated sheep anti mouse igg antibody
1
Centromere Protein Library Construction
2
Western Blot Analysis of IGF-1R Signaling
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Tissue lysates were isolated using RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.5% Na-deoxycholate, 1% NP-40, 0.1% SDS) supplemented with protease inhibitors (Roche, Indianapolis, IN) and phosphatase inhibitors (Sigma). Then, 20 μg of the protein was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes, were immunoblotted using antibodies against IGF-1Rβ, phospho-IGF-1Rβ, SMAD2/3, phospho-SMAD2/3 (Cell Signaling, Danvers, MA, Cat. No. 3027, 3918, 8685P, and 8828 respectively), or β-actin (Chemicon, Billerica, MA, Cat. No. MAB1501). Full scan of western blots are available in Supplementary Fig. 25 . Antibodies were diluted in a ratio of 1:200. Proteins of interest were detected with horseradish peroxidase-conjugated sheep anti-mouse IgG antibody (GE Healthcare, Uppsala, Sweden) and visualized with the Pierce ECL Western blotting substrate (Thermo Scientific, Rockford, IL), according to the provided protocol.
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