Firstly SARS-CoV-2, RBD (1 μg/ml) was incubated with the tested compounds at 37 °C for 2 h then in these proteins was added in well plate overnight at 4 °C and blocked with 2% fat-free milk in PBST for 2 h at 37 °C. 100 μl of sACE2protein was added to the plates and incubated for 2 h at 37 °C. After four washes, the bound protein was detected using hACE2-specific goat antibody (0.5 μg/ml, R&D system) for 2 h at 37 °C, followed by incubation with HRP conjugated anti-goat IgG antibody (1:5,000, Thermo Fisher Scientific) for 1 h at 37 °C. The reaction was visualized by addition of substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO) and stopped by H2SO4 (1 N). The absorbance at 450 nm (A450) was measured by an ELISA plate reader (Tecan, San Jose, CA) [68 (link),69 ].
Hace2 specific goat antibody
Manufactured by R&D Systems
Sourced in United States
HACE2-specific goat antibody is a laboratory reagent designed for use in research applications. The antibody specifically binds to and detects the HACE2 protein, which is a human protein with a role in cellular processes. The antibody can be used in various immunoassay techniques to identify and quantify the HACE2 protein in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
Elisa plate reader, by Tecan (2 mentions)
Ace2 protein, by Abcam (1 mentions)
Axio observer inverted microscope, by Zeiss (1 mentions)
Prolong with dapi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
3 protocols using hace2 specific goat antibody
1
SARS-CoV-2 RBD Binding Inhibition Assay
2
SARS-CoV-2 RBD-ACE2 Interaction Assay
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SARS-CoV-2 RBD (Abcam, UK) (1 μg/mL) was incubated with Biobran at 37 °C for 2 h, and the mixture was added to a 96-well plate, incubated overnight at 4 °C, and then blocked with 2% fat-free milk in phosphate-buffered saline with Tween® detergent (PBST) at 37 °C for 2 h. Diluted ACE2 protein (Abcam, UK) was added to the plates and incubated again at 37 °C for 2 h. After four washes, bound protein was detected using hACE2-specific goat antibody (0.5 μg/mL, R&D system, McKinley, MN, USA) that was incubated for 2 h at 37 °C, followed by the incubation of horseradish peroxidase (HRP) conjugated anti-goat IgG antibody (1:5000, Thermo Fisher Scientific, Dreieich, Germany) for 1 h at 37 °C. The reaction was visualized by adding the substrate 3,3′,5,5′-Tetramethylbenzidine (TMB) (Sigma, St. Louis, MO, USA) and stopped using H2SO4 (1 N). An ELISA plate reader (Tecan, San Jose, CA, USA) was used to measure absorbance at 450 nm and thereby estimate the change in RBD–ACE2 complex formation.
3
Immunofluorescent Staining of hACE2
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To stain for surface-expressed hACE2, BHK21-hACE2 or the control BHK21 cells seeded onto fibronectin-coated glass coverslips were incubated with 0.4 μg/mL of hACE2-specific goat antibody (#AF933, R&D systems) at 4°C in media containing 25 mM HEPES. Next, cells were washed with cold PBS, fixed with 4% paraformaldehyde, and blocked with buffer (2% (w/v) bovine serum albumin, 5% (v/v) glycerol, 0.2% (v/v) Tween20 in Ca2+/Mg2+-free PBS). Secondary donkey AlexaFluor 594-conjugated anti-goat IgG (#A32758 Invitrogen) was used to detect the hACE2 signal. Coverslips were mounted in Prolong with DAPI (Invitrogen) and imaged on an Axio Observer inverted microscope (Zeiss).
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