An amount of 24 μg of pcDNA3.1(+)-Thy1.1 plasmid was transfected into CHO.K1 cells (10 mm Petri dish, 80% confluent) using the lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's recommendation. The following day, cells were plated into a 96-well plate (5 × 105 cells/well). Two days after transfection, an antibody binding ELISA was performed. The cell supernatant was discarded, and either anti-Thy1.1 IgE, IgG2a, or IgG1 were added in serial dilutions. After incubation at room temperature for 1 hour, goat anti-mouse IgE-HRP conjugate (Southern Biotech, 1:4,000) or goat anti-mouse IgG Fc-HRP (Jackson Immuno Research 1:5,000) in 1:1 1% BSA phosphate-buffered saline with Tween 20 (PBS/PBST) were added for 45 minutes at room temperature. Immunoreactivity was visualized with TMB Stabilized Chromogen (Invitrogen). Reactions were stopped after 15 minutes with 0.5 mol/L H2SO4, and absorbances were read at 450 and 620 nm. All samples were tested in duplicate.
Goat anti mouse ige hrp conjugate
Manufactured by Southern Biotech
Goat anti-mouse IgE-HRP conjugate is a laboratory reagent used for the detection of mouse immunoglobulin E (IgE) in various immunoassay applications. It consists of goat-derived antibodies specific to mouse IgE that are conjugated to the enzyme horseradish peroxidase (HRP).
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Lab products found in correlation
Tmb stabilized chromogen, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igg fc hrp, by Jackson ImmunoResearch (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse ige, by Southern Biotech (1 mentions)
Maxisorp plate, by Thermo Fisher Scientific (1 mentions)
2 protocols using goat anti mouse ige hrp conjugate
1
Thy1.1 Antibody Binding ELISA in CHO Cells
2
Quantification of Mouse IgE Antibodies
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96-well MaxiSorp plate (ThermoFisher Scientific) was coated with 0.15μg/ml of goat antimouse IgE in PBS (Southern Biotech, 100μL/well) overnight at 4°C. Blocking was done with 1% BSA in PBS for 1 h at 37°C. Next, supernatants containing IgE antibodies were added in serial dilutions. Initially, before serial dilutions, 2C6 hybridoma supernatant was diluted 4x, whereas FreeStyle293 supernatant containing anti-Thy1.1. IgE was initially diluted 2x with cell culture medium. Mouse IgG1 kappa was used as negative control (clone P3.6.2.8.1). Mouse IgE (Bio-Rad cat# PMP68) was used for the standard curve. The incubation was performed at room temperature for 1 h. Finally, goat anti-mouse IgE-HRP conjugate (Southern Biotech, 1:4000 in 1:1 1% BSA PBS/PBST) was added for 45 min at room temperature. Immunoreactivity was visualized with TMB Stabilized Chromogen (Invitrogen). The reaction was stopped after 15 min with 0.5 M H2SO4 and absorbances were read at 450 nm and 620 nm.
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