Frozen mouse tissues were sonicated in lysis buffer, and the supernatants were collected and used for ELISA. The concentrations of A1-7 and ACE2 were determined using the A1-7 ELISA kit (Cusabio Biotech, Wuhan, China) and the SensoLyte 390 ACE2 Activity Assay Kit (AnaSpec, Fremont, CA, USA), respectively, according to the manufacturer's instructions.
Sensolyte 390 ace2 activity assay kit
Manufactured by AnaSpec
Sourced in United States
The SensoLyte 390 ACE2 Activity Assay Kit is a laboratory tool designed to measure the enzymatic activity of Angiotensin-Converting Enzyme 2 (ACE2). The kit provides the necessary reagents and protocols to quantify ACE2 activity in biological samples.
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10 protocols using sensolyte 390 ace2 activity assay kit
1
Quantification of A1-7 and ACE2 in Frozen Mouse Tissues
2
Quantifying ACE2 Enzyme Activity
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ACE2 activity was evaluated using SensoLyte 390 ACE2 Activity Assay Kit (ANASPEC; cat# 72,086) according to the manufacturer’s protocol. 10 ng or 100 ng of ACE2-Fc and ACE2mod-Fc samples were compared blank control. Measurement of product formation (fluorogenic peptide cleavage) as a function of time was taken every 10 s.
3
Quantifying ACE2 Protein Expression
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For confirmation of the
presence of ACE2 protein in the ACE2-expressing cells, ACE2-expressing
HEK293T and HEK293T control cells were lysed and the activity of the
ACE protein was assessed by the SensoLyte 390 ACE2 Activity Assay
Kit (AnaSpec, USA) according to the manufacturer’s instructions.
The fluorescence (excitation 330 nm, emission 390 nm) of the final
product was read within 5–35 min using a plate reader. The
expression of ACE2 protein in cells was also confirmed by Western
blot analysis, as described above, using a rabbit monoclonal ACE2
antibody (ab239924; 1:1000; Abcam, USA). For FACS analysis, the cells
were seeded in a 6-well plate. Then the cells were detached from wells
by PBS and incubated with ACE2 primary antibody (ab239924; 1:1000;
Abcam, USA) for 1 h on ice, washed with PBS and then incubated with
antirabbit fluorescently conjugated secondary antibody (Alexa Fluor
647 nm) for 1 h on ice. The cells were then washed with PBS, filtered
with 0.45 μm filters, and resuspended in PBS with 2% FBS. The
fluorescence signal (APC filter) of cells was measured by a FACS machine
(LRS-II) and analyzed by the FlowJo software.
presence of ACE2 protein in the ACE2-expressing cells, ACE2-expressing
HEK293T and HEK293T control cells were lysed and the activity of the
ACE protein was assessed by the SensoLyte 390 ACE2 Activity Assay
Kit (AnaSpec, USA) according to the manufacturer’s instructions.
The fluorescence (excitation 330 nm, emission 390 nm) of the final
product was read within 5–35 min using a plate reader. The
expression of ACE2 protein in cells was also confirmed by Western
blot analysis, as described above, using a rabbit monoclonal ACE2
antibody (ab239924; 1:1000; Abcam, USA). For FACS analysis, the cells
were seeded in a 6-well plate. Then the cells were detached from wells
by PBS and incubated with ACE2 primary antibody (ab239924; 1:1000;
Abcam, USA) for 1 h on ice, washed with PBS and then incubated with
antirabbit fluorescently conjugated secondary antibody (Alexa Fluor
647 nm) for 1 h on ice. The cells were then washed with PBS, filtered
with 0.45 μm filters, and resuspended in PBS with 2% FBS. The
fluorescence signal (APC filter) of cells was measured by a FACS machine
(LRS-II) and analyzed by the FlowJo software.
4
Peptide Inhibitors of ACE2 Binding
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Six DX600-derived peptides, named NOTA-PEP1 to NOTA-PEP6 (cyclic vs. noncyclic, with triglycine, aminocaproate, and polyethylene glycol linkers), were studied using a commercially available ACE2 inhibition assay according to the manufacturer’s instructions (SensoLyte 390 ACE2 Activity Assay Kit *Fluorimetric*, AS-72086; AnaSpec). Each peptide inhibitor was first tested at 4 concentrations. Initial velocities were determined relative to the inhibitor free reaction. Subsequently, IC50 values were derived from nonlinear fits of saturation curves of a 6-point dilution series of peptide inhibitors.
5
Quantification of ACE2 Activity
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Measurement of ACE2 activity (SensoLyte 390 ACE2 activity assay kit; AnaSpec, USA) was conducted in accordance with the supplied manual. Briefly, cells were incubated for 15 min at 4 °C and then centrifugated at 20,000 × g for 10 min. The supernatants were collected and stored at − 80 °C for ACE2 activity measurement. Total protein concentration was quantified using the Bradford protein assay.
6
Quantifying ACE2 Activity in Mouse Brain
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ACE2 activity in mice brain was measured by a SensoLyte 390 ACE2 activity assay kit (AnaSpec) with Mc-Ala/Dnp fluorescence resonance energy transfer peptides as described [27 (link)]. The fluorescence of Mc-Ala was monitored at excitation/emission 330/390 nm. Reactions were performed in duplicate in 96-well, clear, flat-bottom polystyrene microplates at a final volume of 100 μL. Specificity was confirmed using DX600, a specific ACE2 inhibitor (AnaSpec).
7
ACE2 Activity Assay Protocol
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The ACE2 activity was assessed with SensoLyte 390 ACE2 Activity Assay Kit (Cat. 72086; Anaspec) by following the manufacturer's instructions. A total of 50 µg of proteins from each tissue of interest was analyzed, and fluorescence was measured after 30 minutes' reactions at Ex/Em = 330 nm/390 nm.
8
ACE and ACE2 Activity Assay in Tissues
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Lung and kidney tissues were lysed in Component C Buffer in SensoLyte 390 ACE2 Activity Assay Kit (Cat. 72086; Anaspec Inc., Fremont, CA) and total proteins were extracted and analyzed in ACE and ACE2 activity assay and western blotting. All these procedures were conducted by following the manufacturer's instructions with minor modifications. Briefly, tissues were homogenized in Component C Buffer containing 0.5% (volume/volume) Triton-X 100 with a Polytron homogenizer at 15,000 rpm for 1 minute, followed by incubation for 15 minutes at 4°C. Tissue lysates were centrifuged for 10 minutes at 1000 ×g at 4°C and the supernatant fractions were collected, aliquoted, and stored at −80°C until analyzed by ACE and ACE2 protein activity assays and western blotting. Protein concentration was determined by using a Pierce BCA Protein Assay Kit (Cat. 23225; Pierce Biotechnology, Rockford, IL).
9
ACE and ACE2 Activity Assays
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ACE and ACE2 activities were measured using the ACE activity assay kit and SensoLyte390 ACE2 activity assay kit (Anaspec, Fremont, CA, United States), respectively, according to the manufacturer’s instructions with some modifications. Briefly, rhACE solution dissolved in deionized water was used for analysis. ACE activity was measured in a reaction system with 50 μl of the sample and 50 μl of the ACE substrate solution with or without BAC (10–4 to 10–2 μM). ACE2 activity was assayed in the same manner as ACE activity. All assays were performed every 10 s for 30 min at 37°C using a Varioskan LUX fluorescence microplate reader (Thermo Fisher Scientific, Waltham, MA, United States). The autofluorescence value in each assay was subtracted from the measured values to generate the final results. The relative fluorescence unit of each sample was normalized to the corresponding total protein concentration, which was measured using a Pierce BCA protein assay kit (Thermo Fisher Scientific).
10
ACE2 Activity Measurement in Mouse Brain
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ACE2 activity was measured using the SensoLyte 390 ACE2 activity assay kit (AnaSpec, CA, USA) according to the supplied manual. Mice were injected i.c.v. with vehicle or DIZE, and brains were removed 1 h later. Brain slices were prepared in the following manner. The excised brains were sectioned into the hippocampus, cerebral cortex, prefrontal cortex, and amygdala using a brain slicer (Muromachi Kikai) and thoroughly homogenized in 150 µL of assay buffer (Component C buffer) provided with the kit. Homogenized samples were incubated for 15 min at 4 °C. After centrifugation (20,000 × g, 10 min, 4 °C), 120 µL of the supernatant was collected and used as samples. The brain samples were reacted with ACE2 substrate for 30 min under light-shielded conditions at room temperature, and after addition of reaction stopper solution, ACE2 activity was measured by measuring fluorescence intensity (excitation: 330 nm /fluorescence: 390 nm) on a SoftMax Pro (Molecular Devices, CA, USA). Total protein concentration was quantified by the Bradford protein assay and used to normalize ACE2 activity.
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