Streptavidin Coated pre-blocked plates (Thermofisher, 15124) were washed 3 times with 200 μL of phosphate buffered saline with 0.1% tween (PBS-T) prior to coating with 100 μL of biotinylated anti-ApoA-I antibody (abcam, ab27630) diluted 1:1400 in PBS-T. Plates were incubated at 37 °C for 2 hours and subsequently washed 6 times with PBS-T. Plasma samples diluted 1:200 in PBS with 0.05% casein (PBS-C) was added to the wells and incubated at 37 °C for 30 min. Following 6 washes with PBS-T, 100 μL of horseradish peroxidase (HRP)-conjugated anti-Human IgG (abcam, ab7153) was added at a dilution of 1:4000 in PBS-C and incubated for 30 min at 37 °C. After a final 6 washes with PBS-T, 100 μL of room temperature tetramethylbenzidine (TMB) (Rockland Immunochemical) was added and allowed to react for 30 min at room temperature in the dark. The reaction was stopped using 100 μL of 0.5 M H2SO4 and absorbance was measured at 450 nm (Biotek, Synergy Hybrid Reader). All samples were analyzed in duplicate.
Ab27630
Manufactured by Abcam
Ab27630 is a monoclonal antibody that binds to the human complement component C3. It is suitable for use in applications such as ELISA, immunohistochemistry, and Western blotting.
Automatically generated - may contain errors
2 protocols using ab27630
1
Quantification of Plasma ApoA-I Antibodies
2
Quantifying ApoA-I Binding to ECM Components
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96-well plates were coated with 25 μg/mL of each of the five ECM components under test diluted in PBS. After overnight incubation at room temperature under agitation, plates were washed with PBS and the free surface was blocked with 1% BSA in PBS for 1 h at 37 °C. The plates were then probed with the different ApoA-I proteins, at increasing concentrations (0–100 μg/mL range) for 2 h at 37 °C. After washing with PBS, samples were incubated with biotinylated anti-human ApoA-I antibodies (Abcam ab27630) for 1 h at 37 °C and thereafter with alkaline phosphatase (ExtrAvidin-AP, Sigma) for 30 min at 37 °C. The assay was developed using p-nitrophenyl phosphate (SIGMAFAST™p-Nitrophenyl phosphate tablets, Sigma) and detected by reading the absorbance at 450 nm using an automatic multiplate reader (Thermo Scientific-Multiskan Go).
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