The soluble fraction of crude extracts were diluted in 100 mM carbonate–bicarbonate coating buffer (28.6 mM Na2CO3 and 71.4 mM NaHCO3, pH 9.6) to a final concentration of 10 μg/ml, and 1 μg of soluble protein was used for ELISA analysis. All process was performed in a 96-well immunoassay plate. The samples were blocked with 200 μl of 5% skim milk in PBS buffer (16.34 mM Na2HPO4, 2.68 mM KCl, 0.94 mM K3PO4, 136.9 mM NaCl, pH 7.4) for 2 h and then incubated with 50 μl of 1: 2,000 dilution of rabbit pAb rGA733-2 for 2 h, followed by 50 μl of 1: 2000 dilution of AP-conjugated goat anti-rabbit IgG (Thermo Scientific, USA) for 1 h. 200 μl of p-nitrophenyl phosphate (1 mg/ml; Sigma-Aldrich, USA) was added as a substrate for AP enzyme. The absorbance of the samples at 450 nm was monitored using a VERSA max microplate reader (Molecular Devices, USA).
Ap conjugated goat anti rabbit igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
AP-conjugated goat anti-rabbit IgG is a secondary antibody reagent used in various immunoassays and detection methods. It is produced by immunizing goats with rabbit IgG and then conjugating the antibodies with alkaline phosphatase (AP) enzyme. This conjugated antibody can be used to detect and visualize the presence of rabbit primary antibodies in a sample.
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Lab products found in correlation
P nitrophenyl phosphate, by Merck Group (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
Rabbit anti mouse igg, by Thermo Fisher Scientific (1 mentions)
96 well polystyrene microtiter plate, by Thermo Fisher Scientific (1 mentions)
Rabbit anti human igg, by Thermo Fisher Scientific (1 mentions)
Powerwave x microplate reader, by Agilent Technologies (1 mentions)
2 protocols using ap conjugated goat anti rabbit igg
1
ELISA Protocol for GA733-2 Protein
2
HSET-N and ss-(dN)40 Peptide Binding Assay
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The 6C407 and 3D8 Ab series (1 μg/ml) in phosphate-buffered saline (PBS) were incubated in a 96-well polystyrene microtiter plate (ThermoFisher Scientific; cat# 439454) coated with 10 μg/ml HSET-N peptide or 5 μg/ml ss-(dN)40 antigen, respectively, for 1 h at room temperature. If necessary, Ab proteins were heated for 10 min to 4 h at 30–90 °C. Mouse IgG Abs bound to wells were detected using a rabbit anti-mouse IgG (Rockland; cat# 610-4503) followed by an alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (ThermoFisher Scientific; cat# 31341). Chimeric MH and MC Abs bound to wells were detected using rabbit anti-human IgG (ThermoFisher Scientific; cat# 31142) and rabbit anti-chicken IgY (Dianova; cat# 303-035-008) Abs, respectively, followed by an AP-conjugated goat anti-rabbit IgG. Color was developed by adding p-nitrophenyl phosphate substrate solution (1 mg/ml prepared in 0.1 M glycine, 1 mM ZnCl2, and 1 mM MgCl2, pH 10.3) to each well. The absorbance at 405 nm was read using a PowerWavex microplate reader (BioTek).
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