Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (BN01071)

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BN01071
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (BN01071)
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (BN01071)
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (BN01071)
  • Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric) (BN01071)
€589

Description

Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Fluorometric)

Angiotensin II converting enzyme (ACE2, EC 3.4.17.23), a zinc-based metalloprotease, is part of the renin-angiotensin system (RAS) that controls the regulation of blood pressure by cleaving the C-terminal dipeptide of Angiotensin II to convert it into Angiotensin 1-7. ACE2 is a receptor of human coronaviruses, such as SARS and HCoV-NL63. It is expressed on the vascular endothelial cells of lung, kidney and heart. ACE2 is a potential therapeutic target for cardiovascular and coronavirus-induced diseases. Assay Genie's ACE2 Activity Assay Kit kit will help the research progress in this field. This kit utilizes the ability of an active ACE2 to cleave a synthetic MCA based peptide substrate to release a free fluorophore. The released MCA can be easily quantified using a fluorescence microplate reader. We also provide an ACE2 specific inhibitor that can differentiate the ACE2 activity from other proteolytic activity.The kit can detect as low as 0.4 mU. Our assay kit is simple and can be used in a high-throughput format.

ACE2 activity assay data

Figure: (a) MCA -Standard Curve (0-300 pmol), error bars indicate SD (n=3). (b) Kinetic activity curves using different amounts of ACE2 Positive Control in the assay. (c) ACE2 activity was measured for different types of rat tissue samples (total protein in lung and kidney; 17 µg and 23 µg respectively), and human kidney tissue sample (10 µg total protein) in presence (+ Inhibitor) and absence (-Inhibitor) of ACE2 Inhibitor. (d) Spiked ACE2 activity and inhibition measured in HEK293 cell lysate (total protein: 37 µg). Assays were performed following the kit protocol

ACE2 activity assay kit components

Key Information Description

Product SKU

BN01071

Size

100 assays

Detection Method

Fluorometric (Ex/Em = 320/420 nm)

Species Reactivity

Mammalian

Applications

Detection of ACE2 activity in tissue/cell lysates and enzyme preparations

Features and Benefits

  • Simple one-step reaction
  • Takes only 1-2 hrs
  • Non-radiometric fluorescent detection HTP adaptable

Kit Components

  • ACE2 Assay Buffer
  • ACE2 Dilution Buffer
  • ACE2 Lysis Buffer
  • ACE2 Positive Control
  • ACE2 Substrate
  • ACE2 Inhibitor (22 mM)
  • MCA-Standard (1 mM)

Storage Conditions

-20°C

Shipping Conditions

Gel Pack

USAGE

For Research Use Only! Not For Use in Humans.

ACE2 function

Carboxypeptidase which converts angiotensin I to angiotensin 1-9, a peptide of unknown function, and angiotensin II to angiotensin 1-7, a vasodilator (PubMed:10969042, PubMed:10924499, PubMed:11815627). Also able to hydrolyze apelin-13 and dynorphin-13 with high efficiency (PubMed:11815627). By cleavage of angiotensin II, may be an important regulator of heart function (PubMed:10969042, PubMed:10924499). By cleavage of angiotensin II, may also have a protective role in acute lung injury (By similarity). Plays an important role in amino acid transport by acting as binding partner of amino acid transporter SL6A19 in intestine, regulating trafficking, expression on the cell surface, and its catalytic activity (PubMed:18424768, PubMed:19185582).

ACE2 Key information

Uniprot

Gene name

ACE2

Alternative names

Angiotensin-converting enzyme 2, ACE-related carboxypeptidase, Angiotensin-converting enzyme homolog, ACEH, Metalloprotease MPROT15

Subcellular location

Extracellular region or secreted, cell membrane, cytoplasm

Tissue Specificity

Expressed in endothelial cells from small and large arteries, and in arterial smooth muscle cells (at protein level) (PubMed:15141377). Expressed in lung alveolar epithelial cells, enterocytes of the small intestine, Leydig cells and Sertoli cells (at protein level) (PubMed:15141377). Expressed in the renal proximal tubule and the small intestine (at protein level) (PubMed:18424768). Expressed in heart, kidney, testis, and gastrointestinal system (PubMed:10969042, PubMed:10924499, PubMed:15231706, PubMed:12459472, PubMed:15671045).

Induction

Up-regulated in failing heart.

ACE2 & the coronavirus

Angiotensin-converting enzyme 2 (ACE2) has been determined to be a functional receptor which facilitates the uptake of SARS-CoV-2 into host cells. (Source) ACE2 is expressed in multiple locations and cell types, notably including alveolar epithelial cells, surface enterocytes within the small intestine, and heart and kidney endothelial cells.

Coronavirus entry into susceptible cells is mediated by the S1 subunit of the coronavirus transmembrane ‘spike’ glycoprotein(commonly abbreviated ‘S’). (Source) S1 contains a receptor binding domain at residues 318–510, which recognises and binds to the LYS341 residue of ACE2 with high affinity. (Source) The affinity of CoV-2 is higher than that of CoV. (Source)

Increased binding affinity to human ACE2 has been correlated with an increase in transmission efficiency in previous outbreaks of SARS-CoV. (Source) Antibodies produced against SARS-CoV-2 target the S2 subunit, which is responsible for the fusion of viral and host membranes. Thus, neutralising the S2 subunit inhibits viral entry into vulnerable cells. (Source) Polyclonal antibodies against the S protein of SARS-CoV have been found to significantly attenuate the ability of SARS-CoV-2 to enter cells via human ACE2. (Source)

ACE 2 Activity Assay Kit - Protocol

Step Procedure

Sample Preparation:

Homogenize tissue (~100 mg) or pelleted cells (1-2 x 10^6) with 400 µl ACE2 Lysis Buffer using a Dounce homogenizer, keep on ice for 10 min. Vortex gently for 10 s, and keep on ice for another 5 min. Centrifuge the homogenate at 16,000 x g, 4°C for 10 min. Discard the pellet.

Protein concentration measurement

Transfer the clarified supernatant to a clean pre-chilled tube and keep on ice. Measure the amount of protein in the lysate or purified enzyme using BCA Protein Assay Kit, Reducing Agent Compatible

Assay Procedure:

For Sample (S), add 1-5 μl of lysate into desired well(s) in a 96-well plate. If necessary, dilute the lysate with ACE2 Lysis buffer. For Background Control (BC), add same volume of lysis buffer. For Positive control (PC), add 2 µl of the diluted ACE2 Positive Control into desired well(s). For Negative Control (NC), add 2 µl of the diluted ACE2 Inhibitor to the wells containing Sample and/or ACE2 Positive Control. Adjust the volume of S, BC, NC and PC to 50 μl/well with ACE2 Assay Buffer. Mix well, incubate for 15 min. at room temperature.

MCA-Standard Curve Preparation:

Prepare 25 µM solution of MCA-Standard by diluting 5 µl of 1 mM MCA-Standard with 195 µl of ACE2 Assay Buffer. Add 0, 2, 4, 6, 8 and 10 µl of 25 µM MCA-Standard into a series of wells in a 96-well plate and adjust the final volume to 100 µl/well with ACE2 Assay Buffer. This will generate 0, 50, 100, 150, 200 and 250 pmol/well of MCA Standard respectively. Mix well and measure the fluorescence (Ex/Em =320/420 nm) in an end point mode.

MCA Substrate Mix:

Prepare enough reagents for the number of assays to be performed. For each well, prepare 50 μl of the Substrate Mix:

 

  • 48 μl ACE2 Assay Buffer
  • 2 μl ACE2 Substrate

Mix & add 50 µl of ACE2 Substrate Mix into each of S, BC, PC and NC wells. Mix well.
Note: Don’t add Substrate Mix to the Standard wells.

Measurement:

Measure fluorescence (Ex/Em 320/420 nm) in a kinetic mode for 30 mins to 2 hr at room temperature. Choose two time points (t1 & t2) in the linear range of the plot and obtain the corresponding values for the fluorescence (RFU1 and RFU2). Calculate ΔRFU/ΔT.

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