Wells of a 384-well plate (ThermoFisher) were coated with oxLDL at 50 μg protein/ml in PBS, and blocked with PBS/ 5% milk as described previously (21 (link)). Dilutions of control protein HSA or purified C1q in PBS/1% milk were incubated for 2 h at room temperature. After washing with PBS/0.05% Tween, monoclonal anti-C1q 1H11(24 (link)) (0.5 μg/ml in PBS/1% milk) was incubated in wells for 90 min at room temperature. Wells were washed prior to incubation with HRP-conjugated anti-mouse IgG secondary antibody (1:1,000 dilution; ThermoFisher Scientific) for 45 min at room temperature. The binding assay signal was developed by the addition of substrate TMB (ThermoFisher Scientific). Binding was assessed by measurement of the average absorbance of triplicate sample wells at 450 nm.
Hrp conjugated anti mouse igg secondary antibody
Manufactured by Thermo Fisher Scientific
The HRP-conjugated anti-mouse IgG secondary antibody is a laboratory reagent used to detect the presence of mouse immunoglobulin G (IgG) in samples. It consists of an anti-mouse IgG antibody conjugated with the enzyme horseradish peroxidase (HRP). This conjugated antibody can be used in various immunoassay techniques, such as Western blotting and ELISA, to amplify and visualize the signal from the target mouse IgG.
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Lab products found in correlation
384 well plate, by Thermo Fisher Scientific (1 mentions)
Tmb substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Enhanced chemiluminescence reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Roche (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
2 protocols using hrp conjugated anti mouse igg secondary antibody
1
Quantifying C1q Binding to oxLDL
2
Western Blot Analysis of Dicer and HER2
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The SGC7901 and MGC803 cells were lysed with radioimmunoprecipitation assay buffer containing protease inhibitor (Roche, Basel, Switzerland). Proteins from each sample were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes (Roche, Basel, Switzerland). After blocking with 5% skim milk, the membrane was incubated with primary antibody against Dicer (dilution rate 1:1,000; Abcam, Cambridge, UK), HER2 (dilution rate 1:1,000; Abcam, Cambridge, UK), and β-actin (1:10,000 dilution; Santa Cruz, CA) at 4 °C overnight, followed by incubation with a HRP-conjugated anti-mouse IgG secondary antibody (1:5,000 dilution; Thermo Fisher, New York, NY) for 1 h at room temperature. The relative intensities of protein bands were visualized using an enhanced chemiluminescence reagent (Thermo Fisher, New York, NY) and analyzed using an FluorChem HD2 protein imprinting imaging system (Alpha Innotec, San Leandro, CA).
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